ELUCIDATING HALAL DRUG:
Identification of Drug and Its Raw Material
by DNA-Based Biochemical Method
Seminar I
Tuesday, March 3rd 2010
Author: Raafqi Ranasasmita (Biochemistry)
Supervisor: Prof. Dr. M. Sadikin, D.Sc.
Reviewer: Sri Ujiani, S.Si (Biochemistry); dr. Indra Kusuma (Physiology)
UNIVERSITY INDONESIA
GRADUATE SCHOOL PROGRAMME
BIOMEDICAL SCIENCE MAJOR
CONTENT
Glossary................................................................................................................
Abstract................................................................................................................
Introduction.........................................................................................................
Content
.....Defining Halal................................................................................................
.....Halal Assessment............................................................................................
.....Polymerase Chain Reaction...........................................................................
.... Sequencing mT DNA .....................................................................................
.....Multiplex PCR...............................................................................................
Conclusion...........................................................................................................
Acknowledgement..............................................................................................
Reference.............................................................................................................
Page
ii
ii
1
1
3
4
5
5
9
9
9
GLOSSARY
Halal
Haram
Qur’an (Koran)
Sunnah
Fatwa
Ijma
: Allowed or lawful; halal drug is a range of drug permitted to
consume by Moslem based on Islamic rule
: Prohibited or unlawful; Anything forbid by God, Allah Azza wa
Jalla, to be act upon or consume upon
: Moslem’s holy scripture
: a record of compilation [hadith] of the traditions in the life,
actions, and teachings of Muhammad the Prophet
: a fatwa (verbal and/or written guidance) based on a ijma’ and
qiyas (referring to other case(s) as an analogy) based on cerain
mazhab (school of thinking), issued by a respected authority.
: Ulama’s (Islamic bishop) consensus deciding the Islamic view
on a particular case(s)
ABSTRACT
Without doubt, moslem is the largest religious group in Indonesia. These groups
of consumer have their own moral values of consuming and medicating, which is
widely acknowledge as halal. With these needs, all stakeholder involved is oblige to
fullfill their (moslems) special need. Halal ethics is based on the guidelines stated in the
Qur’an (moslem’s holy scripture) and Sunnah (a record of compilation of the traditions
in the life, actions, and teachings of Muhammad the Prophet). The rapid developement
of technology have made the halal-status assessment of a product, especially in
medicine, became difficult. This paper will review several knowledge and techniques in
assessing the halal status of a product, its raw material and the various processing
involved. The techniques involved are DNA-based assessment e.g. using PCR to
differentiate between halal product and raw material from its counterpart.
Keyword: halal assessment, drug, biochemical techniques, medicine
INTRODUCTION
The moslem population constitute about one quarter of the total world
population and represent a majority in more than 50 countries, including Indonesia
(Saeed 2001). These group have a special moral values guiding them in consuming and
medicating, which is widely acknowledge as halal. Previous report shows that there is a
storng tendency that the preferences in consumption is influence by the culture
(religion) that the consumer(s) value (Tuschinsky 1995; Merz & Yi 2008; Cleveland
2009). This fact is important for people in the area of medicine (physician,
pharmaceutical company, healthcare agencies, etc) to have a suitable approach to gain
trust and attract their moslem client, in using their service. This is a prerequisite to
survive in a healthcare market that is very competitive and better approach should
continuously made to gain and expand more market. The fact that Moslem costumer
becaming more affluent, formation of Moslem trading block, and Islamization
(including in consumtional behaviour) of Moslem-majority countries is several reason
making the providing Halal service and product essential.
The halal rule taken form the Qur’an and Hadith is simple to understand. Yet,
the involvement of technology in processing and manufacturing makes the process of
understanding and impelemting the rule quite challenging. This paper aim to provide
basic knowledge in understanding halal, its requirement in a medical setting (healthcare
agencies and pharmaceutical company), the assessment involving biochemical
techniques, and examine the prospect and difficulties in its application. This review is
an assigment in completion of Seminar I class, Master Program in Biomedical Science,
Faculty of Medicine, University of Indonesia.
DEFINING HALAL
The largest consumer of drug and other medical treatment in Indonesia is
moslems. Many Moslem consumer is now demanding several special needs in medical
treatment that differ from the remaining consumer. One of it is the halal status (halality)
of the product. The increasing to demand halal drug(s) can be seen from the case of
refusal of polio vaccine and controversy of meningitis vaccine, which both vaccine is
haram yet vital.
Halal (derive from Arabic, “halal”) is ethiologically define as permitted (Eliasi
& Dwyer 2002). Halal drug is a range of drug permitted to consume by Moslem based
on Islamic rule. Haram (forbid) is anything forbid by God, Allah Azza wa Jalla, to be
act upon or consume. Truly, both term (halal-haram) is applicable to activity and
thinking that is cary out by a moslem. For achieving clarity and focus in the discussion,
this paper will limit the definition of halal-haram implementation solely on the act of
consumption, especially medicating.
Basically, the halal status of a drug is very easy to determine. Almost every
substance is halal to consume. Only a view susbtance is haram to consume, and Islam
has provide a clear guidance upon it. These rules is stated in the Qur’an (Koran,
moslem’s holy scripture) and Sunnah (the traditions in the life, actions, and teachings of
Muhammad the Prophet). If there is no clear guidance on particular case(s), a fatwa
(verbal and/or written guidance) based on a ijma’ (ulama’s [Islamic bishop] consensus)
and qiyas (referring to other case(s) as an analogy) based on certain mazhab (school of
thinking), issued by the authority (Riaz & Chaudry 2004). The authority in charge can
be individual respected ulama or organized group of ulama (with fatwa issued by fatwa
commissioner). This authority is oblige to have a competence in related knowledge and
respected by the moslem majority. The law of Islam that stated the urge of halal
medication by a Hadith (a record of compilation) of Sunnah stated by prophet
Muhammad SAW, stating, “Merily, Allah SWT does not create a disease unless there is
a cure for it, and Allah SWT invent a drug for each diseases. You (mankind) are oblige
to medicate and avoid the uses of haram medication (Riwayah Abu Darda). According
to the Qur’an, drug that is haram to consume is a product and its derivate of swine,
blood, animal slaughtered in the name other than Allah (Al - Baqarah :172-173, AlMaidah-3, Al-An’am:145), alcoholic beverages or khamr (Al - Baqarah: 219). Other
materials are considered haram as mentioned in Hadits but some are of these materials
are debatable among different mazhabs (school of thoughts) and require a fatwa (Riaz &
Chaudry 2004; Nasir & Pereira 2008; AIFDC ICU 2008). The other material mentioned
are bird of prey, beast animal, disgusting animal, and animal that is forbid to be killed
(ant and bee). The guide for halal-haram material in this paper will refer to guidance
issued by The Assessment Institute for Foods, Drugs and Cosmetics, Indonesian
Council of Ulama (AIFDC – ICU). Particularly of swine product and its derivate, its is
also categorized as najis, meaning that its contamination in a certain other halal material
convert the status of the material becaming haram. Contaminated appliance by a najis
substance make the appliance require special rinsing method. The particular appliance is
eligible to be used in production of halal product after these rinsing.
If not stated otherwise, guidance and system adopted by Indonesia’s Lembaga
Pengkajian Pangan, Obat dan Makanan—Majelis Ulama Indonesia (LPPOM—MUI) or
The Assessment Institute for Foods, Drugs, and Cosmetics, Indonesian Council of
Ulama (AIFDC—ICU) regarding halal-haram is the one refered in this paper. The
choice is due to the wide acknowledgement of: (1) their reputation by overseas halal
certifier bodies and overseas companies, (2) their system, by trying to accomodate the
views of four major Sunni mazhab, so their decision express the views of majority of
Moslems, (3) their good scientific-based halal assessment, by equipping their Institution
with halal auditor from academician, (4) status, which they represent certifier bodies
from countries with the greates number of moslem in the world. With that reputation
and the author personal experiences, they are the right choice as a main refference of
halal requirement in this paper.
HALAL ASSESSMENT
The detection of haram material in a product is essential, because there is a
chance of contamination by lack of halal-knowledge in manufacturer, fraud, incorect
processing or simply poor traciability of doubtfull origin-ingredients. In other case(s),
global trading have make the distribution of suspicious product (thus requiring halal
asssessment) became more evident. In the field medicine, the hazard is became greater.
The awareness of producing halal drug is not as promising as it is seen in food
production. Several things that cause this is: (1) a lack of data regarding the potential
market for such poduct, (2) drug is considered vital, which the use of vital drug (with no
other alternatives) yet haram is tolerated in Islam, (3) the consumer awareness is not as
great as expected (there is only few case showing consumer reaction against haram
drug), (4) lack of demand from doctor (and specially, their patient), and (5) short of
knowledge of halal-drug production, especially since this are require highly train and
knowledgeable expert.
In species identification for halal assessment of a material or final product, the
concievable method is based either on protein analysis, DNA analysis (Nau et al. 2008),
and lipid (usually fatty acid) analysis (Chin et al. 2009). Several method discuss
elsewhere can either detect one biomolecule alone (such as PCR for DNA; HPLC,
electrophoretic focusing, and ELISA for protein) or can detect different biomolecule
(such as GC-MS for various lipid and protein), as describe in Table 1 (Zhang et al.
2007). The understanding of the nature of each product (and the target molecule) is
esential, and this is the task of scientist. An ulama with background education solely on
religion (such as hadith) is unable and should not be authorize to determine halal status
alone. They must collaborate with respective scientist and build communication among
them. It is a scientist job to provide understanding of work involved in assessing halal
and the nature of the object of asessment. Reciprocally, it is an ulama job to gave the
scientist a religious perspective on such case(s), and gave final decision of the halal
status of a product or material.
Table 1 Common speciation methods used in fish and meat products, their advantages
and disadvantages (Aursand et al. 2003).
Most paper cited in this review intended not solely to detect presence of haram
material. These paper is more interested in detecting the contaminant (unallowed
mixture) of a product which broke the ethics in food production (food adulteration). The
other concer of food adukteration is due to the potential of disease-spreading species,
such as aviani nfluenza virus, bovine spongiform encephalopathy (BSE) and foot and
mouth disease (FMD) in chicken, cattle, pig, respectively (Bai et al. 2009). Selected
paper are took account for reviewing by the author because its ability to detect the
presence of certain raw material(s) which is similar to drug production. The pork and
pork-derived product contamination is the focus of this review since these substance is
both haram and najis, and also because their contamination brought greater concern
compared to contamination of other haram substance. PCR-based method, especially
multiplex PCR will be elucidate more thoroughtly because the alternative method is
much more familiar by general audiences. Method described can be applied solitary or
in adjacent with others.
DNA HYBRIDIZATION
Initial studies using DNA to detect meat species used relatively simple methods.
Labelled DNA probes were hybridized to samples of genomic DNA covalently attached
to nylon membranes in a slot-or dot-blot format, as shown in Figure 1. The presence of
different species in admixtures, commercially processed, heated and canned products,
and cattle of different breeds have been demonstrated. The lack of DNA hybridisation
tests are the relatively time-consuming in labelling of the probe may involve
radioactivity. Neverthless, handling and disposal of radioactive probe is a little bit
complicated.
Figure 1 The basic experimental format for testing a sample by DNA hybridization.
Three samples are shown, species X, Y and Z, being tested against a probe prepared
from species Y.
POLYMERASE CHAIN REACTION
When protein became a target molecule for halal assessment, electrophoresis
techniques is very suitable and showed success for milk and fish species identification
in frozen product and cheese, respectively (Nau et al. 2009). The problem that occur is
that the specific protein for detection needs to be preserve as closely as possible to the
native state. When the product is heated or processed by certain other way, protein is
irreversibly denaturated and can no longer be examined with techniques suitable for
their natives state. DNA analysis is considerably more suitable because DNA stability
toward physical and evironmental conditions and because of its high species-specifity.
Tests are based either on restriction fragment length polymorphism (RFLP), or using
specific primers targeting DNA segment with sufficient species-to-species variation.
Species-specific polymerase chain reaction (PCR) generally offers many major
advantages as a routine method, since it is relatively simple, fast, highly sensitive,
robust, and cheap.
The factor influential in PCR-testing lies in primer design and DNA extraction
(Nau et al. 2009). Primer design can be complicated if there is only a few sequencing
performed and publish publicly for a certain animal. Avoiding cross reactivity is
performed by aligning sequences of mitochondrial DNA in different species. It is
relatively more abundant in total nucleic acid preparations than nuclear DNA,with the
copy number of the mitochondrial genome exceeding that of the nuclear genome several
fold. Mitochondrial DNA tends to be maternally inherited so that individuals normally
possess only one allele and thus sequence ambiguities from heterozygous genotypes are
generally avoided.The relatively high mutation rate compared to nuclear genes has
tended to result in the accumulation of enough point mutations to allow the
discrimination of even closely-related species. It should however benoted that
mitochondrial DNA also exhibits a degree of intraspecific variability. Care has to be
taken when studying differences between organisms based on single base
polymorphisms (Lockley & Bardsley 2000).
Sequencing mtDNA
The most direct means of obtaining information from PCR products is by
sequencing. Most information thus obtained has been used by amplification of
mitochondrial DNA sequences, generally the cytochrome b gene.Since its use in
determining phylogenetic relationships invertebrates, a wealth of sequence data for this
gene has been documented (Lockley & Bardsley 2000).
Multiplex PCR
The developement of multiplex PCR techniques is to encounter the laborious,
expensive and logistically complex as seen in single-species PCR systems. Until now, a
maximum of four PCR systems could be incorporated into a quantitative multiplex PCR
system. This PCR system is able to detect more than one template in a mixture by
addition of more than one set of oligonucleotide primers. Köppel et al. (2009) have
develop a a multiplex PCR system called AllFleisch, which goes beyond this limit.
Simultaneously, it quantifies the DNA of these seven meat species: beef, pork, chicken,
turkey, horse meat, sheep and goat. In cases when appropriate reference material is
available, the actual meat contents can be determined. Boiled and raw sausages as well
as fresh meats. Köppel et al. (2009) carry DNA extraction using a Wizard Plus MiniPreps ® DNA puriWcation system (Promega, Madison, USA). Primers and probes were
established either during this work and base on an earlier work. PCR analysis were
performed using Rotor Gene 6000® real-time system (Qiagen AG Germany) using
“QuantiTect® Multiplex PCR NoROX Master Mix Kit” (Qiagen AG GmbH, Germany)
containing different primer, as illustrate in Table 2 .
Table 2 Primer sequence, concentration, source, PCR product size and labeling of the
probes of the heptaplex qPCR system AllFleisch for the simultaneous determination of
beef, pork, chicken, turkey, horse meat, sheep and goat (Köppel et al. 2009)
Multiplex PCR systems compared to single PCR systems require less time (all
results at once), produce less mistakes (no taking together of results from diVerent PCR
runs), use less template (template DNA serves for analysis of all species at once) and
require less consumables (this counts specially for expensive DNA polymerases). In
addition, inhibiting effects are often ruled out in relative quantification leading to more
robust results. A further advantage is that only one dilution row for the calibration of the
quantification must be included, ameliorating the relation between calibration reactions
and reactions of the samples. In general, the logistical organization in the laboratory
become simpler because less number of tubes have to be handled. Yet, additionally,
some practical considerations must be noted. As offcial food control agencies search for
unexpected contaminants, a broad spectrum of detectable analytes is a prerequisite.
Furthermore, the law usually tolerates minor contaminations (in halal cases, the
toleration is absolutely zero to haram product). Finally, PCR systems need to be cost
efficient to keep laboratory expenses at a reasonable level. Multiplex PCR systems best
fulfill these requirements. AllFleisch can detect fraudulent ingredient declarations by
detecting non-declared meats from the seven most common species. In those cases
when matrix-adapted reference material is available and corresponds closely to the
sample composition, the quantification of meat proportions is feasible. The validation of
quantitative multiplex PCR systems is limited today by the lack of appropriate reference
material. Therefore, there is an urge to put more effort into the generation of reference
data using further variant material.
Results generated using dilutions of DNA for calibration purposes usually do not
correlate with the product’s recipe. Reasons include the degradation of DNA during
production (due to heat and acids) and storage, and/or the use of animal material with
different DNA contents depending on the type of tissue (e.g., muscle fibers orfatty
meat). Neverthless, in halal assessment, this in not considered a limitation since the
system is only concern on the existence of a species-derived product, not the quantity.
Another concern regarding detection of haram substance is the degradation of DNA
sample due to time (in forensic case[s]) and the very small availibility of the target of
detection-material. This is conquer by the detection of primer suitable for spesific
region of Short mt (mitochondrial) DNA Informative Regions (SMIRs) flanked by
conservative regions, as it is performed by Pereira et al. (2006). They design 17 primer
for conserved regions of SMIRs, by aligning and calculating database of two rRNA and
13 protein coding mtDNA genes in large reference sequence of Mamalia class.The use
of short ampiclon (most SMIRs is less than 200 bp in length) is very useful in detection
of higly degraded sample. The problem is, SMIRs based primer is still a theoritical
approach, waiting for application and the existence of a suitable appliances.
Yet, Aida et al. (2004) have done regular PCR analysis on a conserved region in
the mt cytochrome b (cyt b) gene to identify meats and fats of pigs. They latter cut the
the amplified PCR products with restriction enzyme BsaJI resulting in porcine-specific
restriction fragment length polymorphisms (RFLP). The primers used were CYTb1
(50-CCATCCAACATCTCAGCATGATGAAA-30)
and
CYTb2
(50GCCCCTCAGAATGATATTTGTCCTCA-30). Result is shown in Figure 2.
Figure 2 Electrophoresis analysis of DNA extraction from meat and fat samples. The
figure at the bottom is confront with BsaJIrestriction, which the other (top figure) is not.
M-1 kb plus DNA ladder; (1) = mutton; (2) = beef; (3) = chicken meat; (4, 5, 6 and 7) =
pork; (8) = mutton fat; (9) = beef fat; (10) = chicken fat; (11,12,13 and 14) = lard (Aida
et al. 2004).
Another colleagues, Verkaar et al. (2002) have analyse centromeric satellite
DNA, respectively along with mtDNA by a method called SFLP (Satellite Fragment
Length Polymorphism). It encounter limitation in mtDNA detection based on human
MytC sequence, which have several mismatches relative to the bovine sequence. It also
is an answer unability of most method to discriminate meat of cow compare to other
bovine species. The analysis of satellite DNA is especially relevant for the identification
of animals that are of hybrid origin.
CONCLUSION
The understanding of halal requirement, and the science involved is undoubtly essential.
Yet, the application in the field is much more urging to carry out. Proper understanding
and feasibility studies should be performed cerefully in chosing a particular method for
adopting halal food assessment method for halal drug appraisal. All of this effort is a
way to serve moslem consumer who does not only require a good medication, but they
also require a halal ones. This solely intended to provide a medication that is not only
healing physically, but also bring a “spritual” healing.
REFERENCE
Aida AA et al. 2005. Analysis of raw meats and fats of pigs using polymerase chain
reaction for Halal authentication. Meat Sci 69: 47–52.
Al-Hayani FA. 2007. Biomedical ethics: muslim perspectives on genetic modification.
Zygon 42 (1): 153-162.
[AIFDC ICU] The Assessment Institute for Foods, Drugs, and Cosmetics, Indonesian
Council of Ulama. 2008. General Guidelines of Halal Assurance System. Jakarta:
AIFDC ICU.
[AIFDC ICU] The Assessment Institute for Foods, Drugs, and Cosmetics, Indonesian
Council of Ulama. 2009. General Guidelines of Halal Assurance System. Jakarta:
AIFDC ICU.
Aursand MM et al. 2003. Destructive and non-destructive analytical techniques for
authentication and composition analyses of food stuffs. Trends Food Sci Tech 14:
489–498.
Bai W et al. 2009. A novel common primer multiplex PCR (CP-M-PCR) method for the
simultaneous detection of meat species. Food Control 20: 366–370
Boland MJ. 2002. Aqueous two-phase extraction and purification of animal proteins.
Molec Biotechnol 20: 80-93.
Chassy BM.
2009. Global Regulation of Transgenic Crops. Dalam Kriz AL dan
Larkins BA (ed.), Molecular Genetic Approaches to Maize Improvement: 107-124.
Vol 63. Heidelberg: Springer-Verlag Berlin.
Cleveland M. 2009. Acculturation and consumption: Textures of cultural adaptation (in
press). Int J Intercult Relat.
Eliasi JE, Dwyer JT. 2002. Kosher and halal: Religious observances affecting dietary
intakes. J Am Diet Assoc 101 (7): 911-913.
Karim AA, Bhat R. 2008. Gelatin alternatives for the food industry: recent
developments, challenges and prospects. Trends Food Sci Tech 19: 644-656.
Kocturk TO. 2002. Food rules in the Koran. Scandinav J Nutr 46 (3): 137–139.
Köppel R, Zimmerli F, Breitenmoser A. 2009. Heptaplex real-time PCR for the
identification and quantification of DNA from beef, pork, chicken, turkey, horse
meat, sheep (mutton) and goat. Eur Food Res Technol 230:125–133.
Lockley AK, Bardsley RG. 2000. DNA-based methods for food authentication. Trends
Food Sci Technol 11: 67-77
Nau F et al. 2009. Detection of Turkey, Duck, and Guinea Fowl egg in hen egg
products by species-specific PCR. Food Anal Methods 2:231–238.
Merz MA, Yi H. 2008. A categorization approach to analyzing the global consumer
culture debate. Int Marketing Rev 25 (2): 166-182.
Mian R. 1999. Examining the halal market. Prep Foods 81-85.
Mian R, Chaudry M. 2004. Halal Food Production. Boca Raton: CRC Pr.
Pereira F et al. 2006. Analysis of inter-specific mitochondrial DNA diversity for
accurate species identification. Int Congress Series 1288: 103–105.
Saeed M,
Ahmed ZU, Mukhtar SM. 2001. International marketing ethics from an
Islamic perspective: a value-maximization approach. J Business Ethics 32: 127–142
Verkaar ELC, Nijman IJ, Boutaga K, Lenstra JA. 2002. Differentiation of cattle species
in beef by PCR-RFLP of mitochondrial and satellite DNA. Meat Sci 60: 365–369.
Zhang CL et al. 2007. A TaqMan real-time PCR system for the identification and
quantification of bovine DNA in meats, milks and cheeses. Food Control 18: 1149–
1158.
multiplex PCR
the detection of more than one template in a mixture by addition of more than one set of
oligonucleotide primers.
nested PCR
the primers used in the first round of amplification are either both replaced (nested
PCR) or only one is replaced (semi-nested PCR) for the second and subsequent cycles
of amplification. Increases the sensitivity and specificity of the PCR.
quantitative PCR
a means for quantifying the amount of template DNA present in the original mixture.
Usually achieved by the addition of a known amount of a target sequence that is
amplified by the same primer set but can be differentiated, usually by size, at the end of
the reaction.
real-time PCR
a method for the detection and quantitation of an amplified PCR product based on
incorporation of a fluorescent reporter dye; the fluorescent signal increases in direct
proportion to the amount of PCR product produced and is monitored at each cycle, 'in
real time', such that the time point at which the first significant increase in the amount of
PCR product correlates with the initial amount of target template.
reverse-transcriptase PCR (RT-PCR)
a reaction applied when the target sequence is RNA, such as viral RNA or messenger
RNA. Reverse transcriptase that copies DNA from an RNA template is present in the
first round.
Jenis Teknik
Yang Dipakai
Fingerprinting
Molekul Target
Protein
SDS-PAGE
DNA
RFLP dan hibridisasi probe
Keuntungan
Selalu memproduksi
sebuah hasil. Tidak ada
kesalahan positif.
IEF
Elektroforesis 2D
Pemetaan peptida
Kerugian
Identifikasi
berdasarlan
perbandingan
standar
Tidak berguna
campuran
target
terdegradasi
harus
dengan
untuk
dimana
telah
RAPD
Penggunaan sekuen konsensus:
SSCP & RFLP
Pengenalan
Target
Teknik
imunologi
menggunakan
antobodi spesifik
Penggunaan sekuen spesifik
PCR
Hibridisasi blot
Target yang lebih kecil
hanya
terpengaruh
sedikit
oleh
pemrosesan
Intepretasi lebih mudah
Keharusan
untuk
membuat sistem untuk
tiap spesies
Kerap terjadi reaksi
silang dengan spesies
yang sekerabat
Penerapan proses produksi obat yang halal diperkirakan akan meningkatkan penerimaan
konsumen terhadap produk tersebut, yang akan berdampak pada peningkatan penjualan.
Hal ini dikarenakan adanya kecenderungan dimana konsumsi turut dipengaruhi oleh
budaya (agama) yang dianut oleh konsumen (Merz & Yi 2008; Tuschinsky 1995;
Cleveland 2009). Sayangnya, tren minat untuk menjamin agar proses produksi
berlangsung halal dalam industri obat tidak sepesat yang sebagaimana yang terlihat
pada industri makanan-minuman. Ada beberapa hal yang menyebabkan hal ini, antara
lain belum adanya data mengenai besaran pasar dan potensi pasar obat halal. Ini
menyebabkan rendahnya minat produsen karena belum melihat potensi keuntungan
yang akan didapatkan dengan memproduksi obat halal (yang mungkin akan
meningkatkan biaya produksi). Hal lain yang diduga menyebabkan hal ini terjadi adalah
pandangan umum dimana obat dianggap sebagai produk yang vital (baca:
mempengaruhi hidup-mati) sehingga kemungkinan ketidakhalalan sebuah obat
dikesampingkan dengan alasan kegawatan obat tersebut. Disamping kedua hal itu,
rendahnya permintaan konsumen dan dokter akan obat halal membuat tidak adanya
tekanan terhadap produsen untuk menginisiasi produksi obat secara halal. Rendahnya
permintaan dari kedua konsumen ini antara lain kekurang pengetahuan akan bahan dan
proses produksi yang mampu menyebabkan sebuah obat menjadi haram dikonsumsi.
Apalagi, industri obat adalah industri yang melibatkan teknologi tinggi dan bahan
mentah beragam yang memerlukan keahlian tersendiri untuk memahaminya. Tulisan ini
akan berusaha menyoroti kekurangan pada bagian terakhir, yakni minimnya
pengetahuan menganai aspek kehalalan obat herbal.
HALAL ASSESSMENT
Berdasarkan panduan Al-Qur’an dan Sunnah, sebenarnya sangat mudah untuk
menentukan kehalalan suatu obat. Obat-obatan ini setidaknya harus memenuhi 3 aspek
terkait, yakni:
1. Tidak terbuat dari bahan haram (untuk obat dalam).
2. Tidak terbuat dari bahan yang najis (obat luar dan dalam).
3. Tidak terkontaminasi oleh bahan haram (dalam proses produksi, penyimpanan, dan
distribusi).
Pada obat-obatan yang berasal dari tanaman, semua tanaman halal untuk dikonsumsi,
kecuali tanaman yang memiliki efek samping merugikan, seperti beracun. Obat herbal
termasuk sediaan kering dan sediaan galenik (ekstrak, minyak atsiri, infusi atau larutan
tanaman dll) dapat dikatakan halal (Taylor 2001). Ada asAsumsi bahwa obat herbal
berstatus halal telah mendorong konsumsi obat ini terkait keterikatan budaya (Merz &
Yi 2008; Tuschinsky 1995; Cleveland 2009), meski perlu penelaahan lebih lanjut.
Namun yang perlu diwaspadai adalah pesatnya perkembangan ilmu pengetahuan dan
teknologi (terutama manufaktur) membuat penilaian mengenai status kehalalan menjadi
tidak mudah. Hal ini ditambah lagi dengan terjadinya kenyataan perdagangan bebas
dimana proses produksi bahan mentah dan obat terjadi pada daerah yang berjauhan,
dengan kondisi lingkungan yang berbeda. Hal lain yang menjadi kritis adalah pada
aspek proses produksi, pengemasan, penyimpanan dan distribusi yang mampu
menjamin dicegahnya kontaminasi silang bahan haram ke dalam obat herbal yang halal.
Sediaan kering tanaman obat dapat dikatakan halal untuk dikonsumsi, asalkan tidak
terdapat pemrosesan lebih lanjut (selain proses pengeringan) dan tidak tercemari oleh
bahan-bahan yang bersifat najis (kotor). Obat herbal yang berasal dari terkadang
memerlukan pemrosesan lebih lanjut untuk meningkatkan khasiatnya. Setidaknya ada 3
titik kritis yang menentukan kehalalan obat, yakni proses dan bahan isolasi melalui
ekstraksi, proses dan bahan fermentasi dan penggunaan bahan pendukung (eksipien).
Upaya ektraksi bahan aktif dapat dilakukan dengan penggunaan pelarut, antara lain
alkohol. Alkohol sebagai salah satu bahan yang menyebabkan efek serupa khamr, yakni
memabukkan, memiliki ketentuan khusus dalam penggunaannya. Majelis Ulama
Indonesia sendiri memperbolehkan pemakaian etanol sebagai pelarut apabila dalam
produk akhir tidak terkandung residu alkohol. Alkohol yang digunakan pun tidak boleh
merupakan produk samping industri minuman keras (AIFDC ICU 2008; AIFDC ICU
2009).
Selain melalui proses ekstraksi, obat herbal terkadang didapatkan melalui proses
fermentasi. Hal ini antara lain dapat dilihat pada proses pembuatan jus mengkudu
(Morinda citrifolia). Proses fermentasi yang terlalu lama dapat menyebabkan kadar
alkohol meningkat hingga mencapai taraf yang memabukkan, sehingga tergolong
haram. Selain itu, tujuan awal dari pembuatan jus tersebut pun perlu ditelaah, apakah
untuk meningkatkan zat aktif dari mengkudu atau sekaligus untuk mendapatkan efek
minuman keras. Apabila diniatkan untuk membuat minuman keras, hal ini tentu
dilarang dan menjadikan jus mengkudu tersebut haram hukumnya.
Salah satunya hal lain yang menentukan kehalalan proses produksi obat terkait
dengan penambahan bahan-bahan farmasetik, yakni bahan tambahan (bukan obat) yang
diracik bersama obat membentuk produk farmasetik. Bahan-bahan tersebut bisa berupa
substansi pembasah, gelidan, bufer, emulsifier, pewarna, perisa, pemanis, pengisi tablet,
pelarut, bahan enkapsulasi, dll. Bahan-bahan ini bisa saja berasal dari bahan mentah
atau proses produksi yang membuatnya menjadi haram. Bahan kapsul, sebagai contoh,
tergolong sebagai bahan yang kritis status kehalalannya. Kapsul diperlukan untuk
mengemas obat herbal sehingga tercapai tujuan yang diinginkan. Kapsul umumnya
terbuat dari gelatin, sementara kebanyakan gelatin berasal dari babi. Produksi gelatin
dunia pada tahun 2007 adalah sebesar 326.000 ton, dengan 46% diantaranya berasal dari
kulit babi, 29.4% dari kulit sapi, 23.1% dari tulang sapi, dan 1.5% dari bagian lain
(Karim & Bhat 2008). Terkadang, obat herbal dalam proses formualsinya menggunakan
campuran tambahan material yang berasal dari hewan, seperti tulang atau kelenjar
hewan. Hal ini pun harus diwaspadai dengan memastikan bahwa hewan tersebut
tergolong halal.
Salah satu hal yang mampu menjadikan diperbolehkannya pemakaian suatu substansi
haram sebagai obat adalah vitalnya obat tersebut dan ketiadaan alternatif pengganti obat
tersebut. Namun, hal ini memerlukan penilaian ilmiah yang cermat dan hati-hati.
Contohnya akan diberikan dalam dua kasus. Kasus pertama adalah pada penggunaan
sodium (natrium) heparin dan kalsium heparin. Salah satu sumber senyawa ini yang
umum adalah yang berasal dari mukosa usus babi. Bahan ini antara lain berfungsi
antikoagulan darah dalam upaya mencegah penyumbatan akibat gumpalan darah yang
menyumbat (penyebab serangan jantung). Ia pun berperan penting mencegah
penyumbatan darah pada saat operasi jantung dan pada saat dialisis darah. Mengingat
pentingnya bahan ini sebagai antikoagulan, ada yang melegalkan (baca: menghalalkan)
penggunaan obat ini meskipun berasal dari bahan haram. Namun, penilaian yang ilmiah
dan sistematis makan menemukan bahwa ternyata ada alternatif bahan yang halal, yakni
yang berasal paru-paru sapi (yang tentunya disembelih atas nama Allah). Dalam kasus
ini, pemakaian sodium heparin yang berasal mukosa usus babi menjadi haram karena
ketersediaan alternatif. Kasus kedua adalah pada pemakaian vaksin X yang
menggunakan enzim tripsin sebagai katalis. Tripsin umumnya digunakan untuk
melepaskan sel vero dari mikrokarrier (biasanya N,N-diethyl amino ethyl) pada proses
produksi vaksin. Tripsin diperoleh dari ekstraksi protease menggunakan asam atau
alkohol dari pankreas mamalia (umumnya babi). Enzim ini sangat umum digunakan
dalam produksi antara lain vaksin polio oral dan vaksin polio inaktif (Martindale 1977;
Parfit 1999). Enzim ini tidak akan terdeteksi pada produk akhir. (Asumsi atas) ketiadaan
residu tripsin pada produk akhir dan pentingnya vaksin tersebut membuat penilaian
umum menganggap halalnya vaksin yang dibuat menggunakan enzim tripsin babi.
Sebenarnya, ada alternatif enzim tripsin yang berasal dari sapi. Namun, pemakaian yang
belum umum (sehingga memerlukan penelitian mendalam) dan berjangkitnya penyakit
BSE (bovine spongiform encephalopathy) yang populer sebagai penyakit sapi gila,
menyebabkan penggunaan enzim tripsin dari sapi menjadi dihindari. Dalam kasus ini,
pemakaian vaksin yang menggunakan tripsin babi dalam proses produksinya menjadi
halal, sepanjang tiadanya alternatif lain. Namun, proses penelitian untuk mencari
alternatif proses produksi yang halal harus terus menerus didorong untuk mengurangi
konsumsi obat haram karena alasan keterpaksaan.
Aspek kehalalan obat juga sangat terkait dengan obat yang berasal dari produk yang
mengalami rekayasa genetik, yang populer disebut GMO (genetically modified
organism). Secara tradisional, pengenalan (onkorporasi) gen asing dilakukan melalui
sistem persilangan atau perkawinan. Proses ini memakan waktu, dan hasilnya pun
memiliki variasi dengan derajat tertentu. Hal ni diatasi dengan teknologi rekayasa
genetika. GMO melibatkan sebuah penyisipan sebuah gen asing (dari sebuah spesies) ke
dalam gen spesies yang berlainan jenis (Al-Hayani 2007; Chassy 2009). GMO pun
dapat berlaku pada sebuah tanaman untuk memperbaiki karakteristik tanaman tersebut.
Umat Islam belum memiliki panduan yang jelas dan global mengenai proses ini. Namun
secara sederhana, sudah ada kesepakatan mengenai haramnya konsumsi produk yang
berasal dari rekayasa genetika dengan menggunakan gen binatang yang haram untuk
dikonsumsi (seperti gen babi). Jelas, obat herbal yang berasal dari tanaman GMO yang
mengandung gen babi dapat dipastikan keharamannya.
Seminar I
Tuesday, March 3rd 2010
MODELLING DMBA-INDUCE MAMMARY CANCER IN MURINE:
Managing Rat and Mouse in Preclinical Trial
Author: Raafqi Ranasasmita
Supervisor:
Reviewer: Sri Ujiani, S.Si; Adrianus, S.Si
UNIVERSITY INDONESIA
GRADUATE SCHOOL PROGRAMME
BIOMEDICAL SCIENCE MAJOR
CONTENT
GLOSSARY
Murine
DMBA
MNU
: related to a genus (mus) or the subfamily to which its belongs and which
includes the common household rats and mice; relating to, or involving
these rodents, especially the house mouse
: Dimethylbenz(a)anthracene, a carcinogen
: N-methyl-N-nitrosurea, a carcinogen
ABSTRACT
Despite numerous in vivo biomedical research in breast cancer had been
performed, there have been notes about variation of outcome. In carcinogen-induced
breast cancer using rodent, these difference can be found in the rate of tumor growth
and number of tumor per group. This phenomenon especially found on group that solely
received carcinogen (with commercial pellet and water given ad lib). Some protocol fail
even failed to achieve tumor growth, compared to other similar protocol. This review
will assess the requirement to achieve an optimum, replicable and predictable outcome
regarding this kind of model. In particular, this paper will emphasize on
dimethylbenz(a)anthracene (DMBA)-induced Sprague-Dawley rats which is preformed
frequently. This model is chosen because its ability to show human mammary tumor
clinical response to drugs, non toxic, and insignificant tendency to alter rat physiology.
Requirements to achieve a good result are the light exposure, environment temperature,
pathogen-free state, diet, age while carcinogen induction, etc. Nevertheless, there is still
much room for improvement of this experimental protocol. This paper will also discuss
about the optimal and maximal analysis of this model. There is also a brief overview of
the advantage and limitation of other rodent experimental model for breast cancer
research.
Keyword: model, Sprague-Dawley, DMBA, breast, rat, advantage, cancer.
INTRODUCTION: PURSUING THE CANCER MIRACLE
John Fritzgerald Kennedy has announced America’s determination in sending
people to the moon in 1960s, and succeeds in achieving it less in a decade. His
successor, President Nixon, have also declared America’s dream in eliminating cancer
in before the next decade (1980s). We can guess the result. He failed to achieve his
mission. Cancer is not an easy project as it is landing people in outer space. So far, there
are many improvements in this area of research such as prolong survival of breast
cancer patient and the reduction of side effect from the therapy. For some tumor such as
childhood leukemia’s, chance of recovery can reach 90%. Still, the magic bullet drug
for cancer is still far from our reach. Breast cancer, that is very common in women, is
no exception of this phenomenon.
Numerous experimental models, both in vivo and in vitro, for breast cancer have
been generated to understand the biological process and therapy in breast cancer
(Vernon et al. 2007). In vitro assay poorly represent real cancer regarding its deficient
of three dimensional structure and lack of stromal cells. Meanwhile, in vivo assay offer
stroma and three dimensional structure possessing genetic and biomarker abnormalities
similar to their human counterparts (Kim et al. 2004). This reason makes in vivo
experimental model is still favorable despite ethical concerns, especially for the use of
large experimental animals.
The ideal animal model should accomplish several things that is similar to
human: (1) histological feature, (2) stages of progression, (3) physiological and
systemic effect, (4) genes and biochemical pathway involved in its initiation and
progression, (6) tumor that reflect the respond of human tumor to certain therapy, (7)
predictable therapeutic efficacy in human clinical assay and (8) had a susceptible
respond to age and reproductive history (Russo J & Russo IH 2003; Céspedes et al.
2006). One of the problems occurs is the limitation of experimental model to understand
the factor influencing breast cancer and also the curing agent. No model is able to fulfill
all aspects of the requirement. Understanding each available model and its suitability is
essential to achieve a good outcome.
SUITABILITY OF MURINE MODEL
Among available model, murine is one of the most popular one. Spontaneous
mammary tumor is frequently observed in long term study using murine, in this case rat.
Murine mammary carcinomas are a good representation due to the complex multi-step
process of mammary cancer that can be stimulated by chemicals, radiation, viruses, or
genetic fact. Comparative studies with the development of the human breast and the
pathogenesis of breast cancer have contributed to validate rodent-to-human
extrapolation (Russo IH & Russo J 1996). This is, of course, limited to several models.
Most importantly, studies in murine can use inbred strains, which will be essentially
homozygous at most alleles. It is able to be reproduce in high frequencies, involving
(frequently) define molecular changes, and allowing expression of increased phenotype
of low penetration allele (Zarbl 2007). Developing murine and protocol that mimic
critical aspects of human neoplasia is instrumental in framing understanding of
carcinogenesis (Vernon et al. 2007). Various strain and types of murine always comes
with several limitations in representing breast cancer condition. Rats are preferred to
mice because it is easier to palpated the tumor that occur, and dissect the body for
further analysis. Further description will refer to rats to describe all explanation.
Exception will be made if other specification in-detail is illustrated.
Chemically induced rat is the best characterize experimental mammary cancer
model for over 40 years. Rats induced by 7,12-dimethylbenz(a)anthracene (DMBA)
developing tumor is one of the most favourable protocol used among these model. This
rat model is choose extensively to examined breast cancer ethiology, prevention, and
treatment of the disease. This is evidence by the reveal of over 1998 manuscripts in the
NCBI PubMed database when “DMBA”, “rat” and “mammary” are used together as
search terms. The other chemical agent that is widely used is N-methyl-N-nitrosurea
(MNU). However, there is no evidence showing that both agents are carcinogenic for
human. There is even no proof at all showing any chemical showing to cause human
breast cancer (Russo J & Russo IH 2003). Yet, the need for an experimental model is
fundamental to answer the biology of mammary cancer, to assess risk on a certain
treatment and to identify agent that is able to reduce the risk and/or growth of mammary
cancer.
Unfortunately, there have been notes about variation of outcome regarding the
rate of tumor growth and number of tumor per group by this model. This phenomenon
especially found on group that solely received carcinogen (with commercial pellet and
water given ad lib). Some research even failed to achieve tumor-growth, compared to
other similar protocol. This paper will explore DMBA-induce rats as a suitable model,
its advantage, limitation and necessary requirement to achieve a standard and optimum
result. In the end, there will also be an explanation about the advantage and limitation of
other experimental model (as a comparison). This model include transplantation model,
allograft model, xenograft model, orthotropically xenograft model, genetically modified
model, SC implanted athymic mice, and metastastic models.
DMBA-Induced Rodents
The DMBA-induce rodent model is a useful experimental model in testing and
validating the effectiveness of a novel treatment strategies and elucidating physiological
responds. It is useful in replicating advances cancer, predicting clinical response to
therapy and reproducing the histology pattern. However, this model is not useful in the
study of metastasis. Unfortunately, metastasis is the main determinants of the clinical
course of the disease, the patient’s survival, and the target of systemic therapy. This is
since DMBA induced spontaneous tumor often fails to metastize after extremely long
latency period (Vernon et al. 2007). Problems occur relating the lack of reproducibility
of various researches using this model (Russo J & Russo IH 2003). The strongest
statement of this condition is stated by Anderson et al. (2002) showing difference result
obtain from two separated studies concerning assessment of 50-Hz MF power-line
frequency exposure effect to the development of tumor. Those studies uses (what is
thought in the first place) a similar protocol, and acquire a statistically different output
and interpretation.
Murine Consideration
Inbred SD (Sprague-Dawley) strain, outbred SD strain, inbred Wistar/Furth
(WF) strain, and inbred Lewis strains were found to be as highly susceptible to DMBA
exposure as the randomly outbred SD strain (i.e., >2 mammary adenocarcinomas/rat
develop). The susceptibility of SD to DMBA-induction is comparable to WF, Fischer
344 (F 344), and Long-Evans (LE) rat. WF rats along with SD, Buffalo/N (Buf/N), and
Lewis rats have the highest tumor development, followed by intermediate strains (F344,
August, and LE rats), then followed by totally-resistant Copenhagen (Cop) rats
(DeAngelo 1978; Isaac et al. 1986; Moore et al. 1988; Russo J & Russo IH 1996; Zarbl
2007). SD strain is preferable compared to other strain due to its susceptibility to
mammary cancer by carcinogen induction (Moore et al. 1988) and its tendency to
develop cancer spontaneously. It is also more susceptible to develop tumor due to
carcinogen induction (Kubatka et al. 2002). The frequent use is making this model well
documented, provides a good comparison as a guideline. Still, there is an inherent
difference on neoplastic response of mammary tissue to DMBA between SD-outbred
rats (substrain) obtain in the US, compared to others in Europe (Anderson et al. 2002).
This is because different breeding may result to a different genetics of rats obtained.
Another limitation of this model lies in its incapability to showed parallel mutagenesis
as found in human (because the disparity of xenobiotic metabolism) and lack of genetic
complexity (due to the use of inbred strain) (Khanna & Hunter 2005).
However, there are relative sensitivities between strain and its susceptibility to
develop cancer by carcinogen induction. As an example, August-Copenhagen-Irish
(ACI) strain is highly sensitive to carcinogenic effect to estrogen and, in contrast, shows
low susceptibility to chemical carcinogen. Cop and Wistar-Kyoto (WKy) strains are
remarkably resistant to the development of both spontaneous and carcinogen-induced
mammary tumors (Zarbl 2007).
Nulliparous rats and rats undergoing pregnancy interruption are more
susceptible to developing carcinomas (Russo et al. 1982; Medina & Smith 1999). This
is making the use of virgin rats a requirement. Mice and rats itself have a different
number of mammary gland. The female rat has six pairs mammary gland, the fifth and
sixth pairs being located in the inguinal region. Female mice, on the other hand, have
five pairs: one cervical, two thoracic and two abdominal-inguinal pairs. There is no
other distinct morphological difference between both species (Russo IH & Russo J
1996). Histopathological and anatomical feature, of normal and DMBA-induced rodent,
are discussed elsewhere (Huggins & Fukunishi 1963; Haslam & Source 1977; Russo
1982; Russo IH & Russo J 1996; Ting et al. 1997; Russo J & Russo IH 2000;
Thompson & Singh 2000; Russo J & Russo IH 2003). The location in and nearby
mammary gland are the most potential area which tumor is found.
Comparison Between DMBA and MNU
DMBA is known to encapsulate (Steele et al. Unknown year), making rats does
not metastize. It is an indirect acting carcinogen (familiarly recall as pro carcinogen)
requiring metabolic intervention to biotransformated it to an active form. This
carcinogen tends to induce high incidence of adenomas and fibroadenomas. This is
making the use of its counterpart, MNU, is much more preferable (Moon 1993).
Premalignant stages, of the disease, are best characterized by both models (Thompson
& Singh 2000). However, DMBA have a greater tendency developing mammary cancer
by oral induction. These models are suitable to assess chemopreventive properties of an
agent (Ip 2006).
MNU does not required metabolic inactivation. Therefore, the use of MNU will
restrain the detection of agents that alter carcinogen metabolism (Steele et al. unknown
year). This chemical agent also showed weakness regarding its different response
compared to human. Gene expression profile of NMU induced Lewis rats showed that
treatment using MNU increases level of expression of cell-proliferation genes (Igf2 and
Igfbp4) in virgin rats, but not in parous rats (Shull 2007). Human shows opposite nature,
which virgin women had a lower risk to develop mammary tumor (showed by cell
proliferation) compared to parous mother.
MNU administration boost ras expression up to 75%, higher to DMBA (only
about 25%). Mutation of ras is extremely rare in human breast cancer, with activation
through amplification occurring in less than 25% of human breast tumors (Clarke 1996).
This characteristic briefly showed that MNU is incapable to mimic molecular event in
human mammary cells.
The administration of MNU by the popular intravenous-route, whether via the
foot vein, tail vein, or jugular vein, requires a certain degree of technical skill. It is also
a time-consuming procedure because the animals have to be anesthetized and/or going
through surgery. The prepared solution must be used as quickly as possible because it
decomposes quickly in an aqueous solution. This is another reason making DMBA is
much more preferable to MNU (Ip 1996). Detail comparison between both experimental
models, is described by Thompson and Singh (2000).
Effect, Benefit, Limitation, and Requirement of DMBA Administration
DMBA as a part of polycyclic aromatic hydrocarbon (PAH) molecules is also
preferable because it is studied frequently, and produces a powerful baseline level of
tumor incidence. DMBA-induced rat models is first preformed in 1961 (Huggins et al.
1961), compared to MNU induced rats in 1975 (Shull 2007). This historical factor and
the enormous amount of research using DMBA, compared to MNU, making the
DMBA-induced mammary cancer is well documented and, thus, prefer. Comparison
between both carcinogen is discussed by others (Russo J & Russo IH 1996; Ip 2006).
Age of rats during induction is essential factor that affect tumor formation.
However, there is a difference recommendation regarding age when carcinogen is
highly susceptible. It is recommended to induce DMBA at the age of 40-60 days old
(Russo J & Russo IH. 2003), which is the early period of vaginal opening and sexual
maturity.
High susceptibility of female rats can be observed in carcinogen application
between postnatal days 40-60 in early puberty, with highly proliferating terminal end
buds (TEBs) in the mammary gland. During the age of 40 and 46 days, rats correspond
to the period when mammary gland exhibits the highest density of extensively
proliferating and differentiating TEBs (Kubatka et al. 2002; Russo J & Russo IH. 2003).
DMBA administration between these ages resulted the highest tumor formation.
Correlation between DMBA dosage, frequency of administration (either single
or multiple times), latency (time for tumor to develop), and tumor growth is described
elsewhere (Steele et al. Unknown year; Carter et al. 1988; El-Bayoumy 1994; Steele et
al. 1994; Russo J & Russo IH 1996; Russo J & Russo IH 2003; Ranasasmita 2008).
Until now, there is no general agreement on the effective dosage. Yet, multiple
administrations of DMBA tend to encourage tumorigenesis much progress as describe
elsewhere. The co-administration of an agent with DMBA could alter tumorigenicity by
changing the pharmacokinetic properties of DMBA (Clarke 1996). It could produces a
potentially confounding results, thus requiring careful consideration.
Oral induction of DMBA resulted a mammary tumor arose form ductal ephitelial
cell with morphological feature similar to human mammary cancer (Manna et al. 2007).
Pathogenesis in human is reflected by this protocol (Jonkers et al. 2007), which
carcinogen exposure is likely to result from oral suplementation, not due to physical
interaction. Blood vessel and lymphatic vessel is also produced by this handling
(Motoyama et al. 2008). This reason makes the oral induction of DMBA reflect
pathogenesis of this disease better, compared to two-stage topical application of DMBA
(with the aid of 12-O-tetradecanoylphorbol-13-acetate [TPA]). Physiological and
anatomical aspect of human mammary cancer is mimics by this protocol. Normal
initiation and progression of cancer imitate human tumorigenesis after carcinogen
induction. Yet, rapid onset of mammary cancer due to carcinogen induction in rodent is
distinct to human because human usually develop cancer in a long span of exposure.
Possible explanation of transformation include less efficient DNA repair, poor control
of genetic stability, immortalization, easiness of rodent cells became immortalized,
and/or alter control of gene expression (Kim et al. 2003). Chemopreventive agent is
suitable to be assessed using this model. This model can also assess effect of a certain
therapy to treat estrogen-receptor (ER) positive mammary cancer (Baylor 2003).
Other Consideration
Temperature, quality and quantity of lighting, reproductive event,
endocrinologic milieu, gaseous contaminant (such as ammonia) are also a significant
experimental variable affecting research. Chronic lung infection, which usually
unexpectedly develops in lab, is an aetology that lower tumor incidences (Jull 1966;
Baker et al. 1979; Russo J et al. 1982; Russo J & Russo IH 1996).
It is recommended to use 12-hour light/dark lighting cycle with an ideal room
temperature between 21°C-22°C. There is a suspicion of seasonal influence for 4
season-climate countries due to circannual oscillations in the pineal melatonin
production (Kubatka et al. 2002).
Diet also had profound impact on rat sensitivity. Consumption of sugar and
starch will promote more tumor progression compared to lactose due to restriction of
energy consumption in lactose-fed rat (Klurfeld et al. 1984). Several research usually
fed rat solely with glucose hours to days before and after i.g. DMBA administration.
This treatment is to compensate toxic effect of carcinogen, such as decreased food
consumption and adrenal necrosis (Carter et al. 1988). High fat diet is suspected to
enhance mammary carcinogenesis (Simon 1991; Welsch et al. 1991; Wynder et al.
1997). However, recent findings (Kimura & Sumiyoshi 2007) showed that high fat,
high-sucrose and high-cholesterol diets accelerate tumor growth and metastasis in
tumor-bearing mice. Unfortunately, this conclusion is achieved using an unpopular
drug-resistant Lewis lung carcinoma (LLC) mouse and C57 BL/6J mice. The choice of
strain making this Kimura and Sumiyoshi conclusion difficult to be extrapolate to
DMBA-induced SD strain. Above all, there is no straightforward conclusion regarding
ideal dietary supplementation supporting ideal level of tumorigenesis.
RODENT MODEL: DIFFERENCES AND ALTERNATIVES
Classification of mammary tumor in rodent is not equivalent to standard human
pathology grades and types. Animal model is proofed to be useful in understanding
tumor initiation and primary tumor growth due to its tendency to develop tumor
frequently. Yet, they are less metastatic compared to human in a variable degree. They
also had some physiological feature that is different compared to human. Rodent does
not bear the same genetic alteration and had higher metabolic rates.
As a small animal, rodent own larger respiratory quotient than human, resulting
a different animal tissue microenvironment. There is also distinctive biochemical
feature, such as longer and much more-actives telomere in rodents that make them
undergo spontaneous immortalization. Knowledge of different experimental model is
necessary to be able to select an appropriate model to support the studies undertaken.
Transplantation Model
Transplantation model is one of the oldest techniques to develop mammary
cancer in rodents. Cells derived from breast cancer are transplanted in situ. There are
several considerations regarding this technique. Special concern should be notice on the
transplantation site. Early pathogenesis of this model differ compared to human because
the lost of several aspects; architectural and cellular complexity (Kamb 2005), tumorstroma interaction, detachment and local invasion, and extravasation.
Allograft models
This model is an advancement of xenograft model because of the elimination
of graft versus host reaction found in xenograft model. It involves syngeneic recipient
rodent with identical background which is cancer-transplanted. Most allograft models
do not show extensive metastatic pattern, with lung cancer models as an exception
(Jonkers et al. 2007). However, genetic heterogeneity (which is a hallmark of cancer in
human) is not retained (Vernon et al. 2007).
Xenograft models
Immunocompromised rodent (nude, SCID, etc) is used as a host for
xenotransplantation of human tumor. The result is mosaic of human cancer cells and
murine stromal cells. This model is beneficial when used to assess the biological
behavior human tumor (especially the complex aspect) and its response to drugs
(Rangarajan & Weinberg 2003; Céspedes et al. 2006; Gutmann et al. 2006). Tumor
develops rapidly by using this approach than the natural event.
Xenograft models can not study the contribution of intact immune system.
Meanwhile, immune system has a profound role in altering the tumor
microenvironment, angiogenic pathway, and tumor survival (Vernon et al. 2007). It also
had limited capabilities to dissect early molecular event. Moreover, human cells used in
this model can not adapt to mouse microenvironment (Jonkers et al. 2007; Gutmann et
al. 2006), (sometimes) also missing cancer cells and stromal cells interactions. The
endrocine responsiveness of several xenograft models has "flip-flopped" from estrogendependent/antiestrogen responsive to estrogen-inhibited/antiestrogen resistant (Clarke
1996).
Breast cancer cell is much more difficult to be transplanted in xenograft rodent
due to difficulties regarding site of implementation, hormone supplementation used, age
and strain of rodent used. The ability of xenograft to accurately predict drug response
efficacy have been disappointing. Most of this limitation is overcome by the
development of orthotropic xenograft models.
Orthotropically Xenograft Models
This model use immunosupressed-mice that are transplanted orthotropically.
Cancerous cells is injected or transplanted surgically to a location which the tumor
naturally comes. This approach mimics the pattern of invasion, dissemination,
metastasis, histology, vascularization of several tumor types, gene expression, and
responsiveness to chemotherapy (Khanna & Hunter 2005; Céspedes et al. 2006). In
drug development, this is a suitable model to reproducing clinical response from several
human tumors. The weakness of this approach lies in inability to examine the initial
phase of tumorigenesis.
Genetically Modified Mouse
Genetically modified (transgenic) mouse (GMM) involved over expression of
oncogenes through mutagenesis experiment. Similar genes and molecular event occur is
triggered in GMM model. This model offer a chance to studied specific causal
relationship in tumorigenesis (especially initiating genetic event) and identifying
synergestic pathway (Céspedes et al. 2006). Another advantage of this model are: (1)
initiating genetics event is known, (2) rodent is immunocompetent, (3) tumor develop
spontaneously in their appropriate tissue compartment and (4) ability to determine the
role of microenvironment in tumor formation and progression (Gutmann et al. 2006).
There are many promotor involved as described elsewhere (Jonkers et al. 2007). This
promoter required hormone-regulated enhancer element. This element is naturally
absent in human. Sometimes, this element sometimes also lead to an unexpected
response (Kim et al. 2003), such as tumor enhanced by pregnancy. Yet, gene mutation
should occur in several regions due to nature of cancer and its pathogenesis.
Histopathological feature of GMM is much more analogous to human compared to
MMTV (mouse mammary tumor virus)-induced and carcinogen-induced rodent. This
model is achieved by two ways, either injection of mutant cell injected to
immunocompromised animal or cross breeding of transgenic rodents expressing PyMT
to others deficient a specific gene (Vernon et al. 2007).
GMM failed to replicate advances cancer since a particular genetic alteration
lead to a different tumor types (compared to humans) and lower the metastatic rates
(Céspedes et al. 2006). The specificity of this model makes it loose it other
characteristic. Compared to carcinogen-induced rat, this model is not suitable for drug
testing because of it own dissimilarity that is contrast to human.
GMM model is less favorable to provide a good baseline through statistical
analysis. This model also failed to replicate specific time window and showing subsets
of cell that is mutation that is impact environmentally. This weakness is overcome by
advancement of the method as described elsewhere (Cardiff et al. 2002; Herzig &
Christofori 2002; Céspedes et al. 2006). Patent law issued to Harvard University and
licensed solely to DuPont Inc perhaps makes this model economically less unfavorable.
Detailed explanation is described elsewhere (Bennett & Wiseman 1997; Recio & Everitt
2001; Borowsky 2007).
SC implanted athymic mice
The most widely used models, athymic mice sub cutaneously implanted by
human tumor, also comes with several problems. This model lack of correlation with
clinical response and occasionally does not respond to drugs that are already used
clinically. The implementation site changes drug sensitivity in a reversible way.
However, it is very helpful in understanding oncogenesis (Céspedes et al. 2006). This
model is widely used in pharmaceutical companies to evaluate drug and continues to be
accepted by the US FDA (Federal Drug Administration).
Metastastic Models
This model is achieved by intra vein injection of breast cancer cell either into the
tail vein (resulting pulmonary metastasis), portal vein (for lung colonization), foot pad
(to invade and enter blood stream), or artery (colonization of liver). Intra cardiac
injection of carcinoma cell results in metastasis to a broader range of organ, but tends to
spread to bone (Céspedes et al. 2006; Fantozzi & Christofori 2006; Vernon et al. 2007).
This process bypass early step of metastasis, so only a partial process of metastasis can
be examined. This became the reason ruling out the use of this model to preclinical trial
testing anti metastatic properties of an active agent (Khanna & Hunter 2005). Detailed
explanation is describes by Cardiff et al. (2002).
CONCLUSION
Regulatory agencies should state a clearer definition of required preclinical
model that represent population that will be treated with the drug candidate. Yet,
economical reason should also be considered to acquire maximum understanding with a
minimum cost. The development of a drug should not only involve a single model and
approach, but used other approach with in depth understanding. This approach should
be used in dissecting other element of tumor.
Choosing the most suitable model is essential for an optimal result. Yet, analysis
is also an instrumental tool. Noninvasive imaging should be performed more frequent.
They are radionuclide, bioluminescence imaging (BLI), fluorosence imaging, magnetic
resonance imaging, positron emission tomography (PET), magnetic resonance imaging,
ultrasound, computer tomography (CT), single photon emission computer tomography
(SPECT), multiphoton microscopy and in vivo flow cytometry will allow non invasive
examination for mammary cancer profiling (Fantozzi & Christofori 2006; Jonkers et al.
2007; Vernon et al. 2007). Data interpretation, attempt to make clinical correlation and
believes in the predictive power of the selected models, must consider all limitation that
is bear by the model. This is intended solely to have an impartial result.
ACKNOWLEDGEMENT
The author would like to thank Angga Delta, Irdham Kusuma, Yudi Rusman, and Joe
for providing the required materials and the kind support of Agung E. Wibowo,
Tarwadi, and Augustijana Kartasasmita while writing this paper. I apologize for the
work of colleagues not describe due to space consideration.
LITERATURE
Alani RM, Silverthorn CF, Orosz K. 2004. Tumor angiogenesis in mice and men.
Cancer Biol Ther 3:498-500.
Anderson LE, Morris JE, Sasser LB, Loscher W. 2000. Effects of 50- or 60-Hertz,
100μT magnetic field exposure in the DMBA mammary cancer model in SpragueDawley rats: Possible explanation for different result from two laboratories.
Environ Health Perspect 108: 797-802.
Baylor P. 2003. The mouse in preclinical trials: transgenic, carcinogen-induced, or
xenograph models — which to use? Breast Canc Res 5 (1):S8.
Bennett LM, Wiseman RW. 1997. Mouse models for breast cancer susceptibility.
Environ Toxicol Pharmacol 4: 283–288.
Borowsky A. 2007. Special considerations in mouse models of breast cancer. Breast Dis
28:29–38.
Cardiff RD et al. 2002. Mouse biology in breast cancer research. Compar Biol Med 52:
12-31.
Carter JH, Carter HW, Meade J. 1988. Adrenal regulation of mammary tumorigenesis
in female Sprague-Dawley rats: Incidence, latency, and yield of mammary tumors.
Canc Res 48: 3801-3807.
Céspedes MV, Casanova I, Parreño M, Mangues R. 2006. Mouse models in
oncogenesis and cancer therapy. Clin Transl Oncol 8: 318-329.
DeAngelo AB, Hudgins WR, Kerby SB. 1978. 7,12-Dimethylbenz[a]anthraceneinduced alteration of mammary epithelial cell RNA-synthetic patterns in rat strains
of high and low tumor incidence. Canc Res 38: 384-389.
El-Bayoumy K. 1994. Evaluation of chemopreventive agents against breast cancer and
proposed strategies for future clinical intervention trials. Carcin 15 : 2395-2420.
Fantozzi A, Christofori G. 2006. Mouse models of breast cancer metastasis. Breast
Canc Res 8:212.
Freedman LS. 1990. Enhancement of mammary carcinogenesis by high levels of dietary
fat: A phenomenon dependent on ad libitum feeding [Corespondence]. J Nat Canc
Inst 83:299-300.
Gutmann DH, Schaedle KH, Shannon KM. 2006. Harnessing preclinical mouse models
to inform human clinical cancer trials. J Clin Invest 116: 847-852.
Haslam SZ, Source HAB. 1977. Histopathogenesis of 7,12-dimethylbenz[a]anthraceneinduced rat mammary tumors. PNAS 74: 4020-4024.
Herzig M, Christofori G. 2002. Recent advances in cancer research: mouse models of
tumorigenesis. Biochim Biophys Acta 1602: 97– 113.
Huggins C, Fukunishi R. 1963. Mammary and peritoneal tumors induced by
intraperitoneal administration of 7,12-Dimethylbenz[a]anthracene in newborn and
adult rats. Canc Res 23: 785-789.
Huggins C, Grand LC, Brillantes FP. 1961. Mammary cancer induced by a single
feeding of polynuclear hydrocarbons, and its suppression. Nature 189: 204-207.
Ip C. 1996. Mammary tumorigenesis and chemoprevention studies in carcinogen-treated
rats. J Mammary Gland Biol Neoplas 1: 37-47.
Isaacs JT. 1986. Genetic control of resistance to chemically induced mammary
adenocarcinogenesis in the rat. Canc Res 46: 3958-3963.
Jonkers J, Derksen PWB. 2007. Modeling metastatic breast cancer in mice. J Mam
Gland Biol Neoplas 12:191–203.
Jull JW. 1966. The effect of infection, hormonal environment, and genetic constitution
on mammary tumor induction in rats by 7,12-dimethylbenz(a)anthracene. Canc
Res 26: 2368-2373.
Kamb A. 2005. What’s wrong with our cancer models? Nat Rev: 161-165.
Khanna C, Hunter K. 2005. Modeling metastasis in vivo. Carcin 26:513-523.
Kim JB, O’Hare MJ, Stein R. 2004. Models of breast cancer: is merging human and
animal models the future? Breast Canc Res 6:22-30.
Kimura Y, Sumiyoshi M. 2007. High fat, high-sucrose and high-cholesterol diets
accelerate tumor growth and metastasis in tumor-bearing mice. Nut Canc 59 (2):
207–216.
KIurfeld DM, Weber MM, Kritchevsky D. 1984. Comparison of dietary carbohydrates
for promotion of DMBA induced mammary tumorigenesis in rats. Carcin 5:423425.
Kubatka P et al. 2002. Variability of mammary carcinogenesis induction in female
Sprague-Dawley and Wistar: Han rats: The effect of season and age. Physiol Res
51: 633-640.
Medina D. 2008. Premalignant and malignant mammary lesions induced by MMTV and
chemical carcinogens. J Mammary Gland Biol Neoplas 13:271–277.
Medina D, Smith GH. 1999. Chemical carcinogen-induced tumorigenesis in parous,
involuted mouse mammary glands. J Nat Canc Inst 91: 967-969.
Moon RC. 1993. Premalignant lesions of the breast: Animal models. J Cell Biochem
17G:65.
Moore CJ, Tricomi WA, Gould MN. 1988. Comparison of 7,12dimethylbenz[a]anthracene metabolism and DNA binding in mammary epithelial
cells from three rat strains with differing susceptibilities to mammary
carcinogenesis. Carcin 9:2099-2102.
Motoyama J et al. 2008. Hyperthermic treatment of DMBA-induced rat mammary
cancer using magnetic nanoparticles. Bio Mag Res Tech 6:2.
Ranasasmita R. 2007. Anticancer activity of Aglaia elliptica Blume leaves ethanol
extract on female rats induced by 7,12-Dimethylbenz(a)anthracene [undergraduate
thesis]. Bogor: Bogor Agriculture University.
Rangarajan A, Weinberg RA. 2003. Comparative biology of mouse versus human cells:
Modelling human cancer in mice. Nature Rev 3:952-959.
Recio L, Everitt J. 2001. Use of genetically modified mouse models for evaluation of
carcinogenic risk: Consideration for the laboratory animal scientist. Comparat
Med 51:399-405.
Russo IH, Russo J. 1996. Mammary gland neoplasia in long-term rodent studies.
Environ Health Perspect 104:938-967.
Russo J, Russo IH. 2000. Atlas and histologic classification of tumors of the rat
mammary gland. J Mammary Gland Biol Neoplas 5: 187-200.
Russo J, Russo IH. 2003. Mammary tumor induction in animals as a model for human
breast cancer. In: Allison MR (eds). The Cancer Handbook. Unknown city:
Nature. p 923-935.
Russo J, Russo IH. 1996. Experimentally induced mammary tumors in rats . Breast
Canc Res Treat 39: 7-20.
Russo J, Tay LK, Russo IH. 1982. Differentiation of the mammary gland and
susceptibility to carcinogenesis. Breast Canc Res Treat 2: 5-73.
Shull JD. 2007. The rat oncogenome: Comparative genetics and genomics of rat models
of mammary carcinogenesis. Breast Dis 28: 69–86.
Simon R. 1991. Evaluating the differential effect of diet on rat carcinogenesis. J Nat
Canc Inst 83: 1261-1262.
Steele VE et al. 1994. Preclinical efficacy evaluation of potential chemopreventive
agents in animal carcinogenesism: Methods and results from the NCI
chemoprevention drug development program. J Cell Biochem 20:32-54.
Steele VE, Lubet RA, Moon RC. Unknown year. Preclinical animal models for the
development of cancer chemoprevention drugs. In: Cancer Chemoprevention.
Vol-2. GJ Kelloff, ET Hawk, CC Sigman, editor. New Jersey: Humana Pr.
Thompson HJ, Singh M. 2000. Rat models of premalignant breast disease. J Mammary
Gland Biol Neoplas 5:409-420.
Ting AY, Kimler BF, Fabian CJ, Petroff BK. 2007. Characterization of a preclinical
model of simultaneous breast and ovarian cancer progression. Carcin 28:130–135.
Vargo-Gogola T, Rosen JM. 2007. Modelling breast cancer: One size does not fit all.
Nature Rev Canc 7:659- 669.
Vernon AE, Bakewell SJ, Chodosh LA. 2007. Deciphering the molecular basis of breast
cancer metastasis with mouse models. Rev Endocr Metab Disord 8:199–213.
Welsch CW, House JL, Herr BL, Eliasberg SJ, Welch MA. 1990. Enhancement of
mammary carcinogenesis by high levels of dietary fat: A phenomenon dependent
on ad libitum feeding. J Nat Canc Inst 82:1615-1620.
Zarbl H. 2007. Toxicogenomic analyses of genetic susceptibility to mammary gland
carcinogenesis in rodents: Implications for human breast cancer. Breast Dis 28:
87–105.