Globalization has made world smaller and it is only possible due to
communication and transportation facilities. In the case of transportation we
cannot ignore revolution in automobile and mechanical engineering
research. Almost all vehicles having internal combustion engine need fuel.
Only crude oil is the source to make all the petrochemicals and mainly fuels,
that’s why it’s also denoted as “Black Gold”. Liquefied Petroleum gas
(LPG), petrol, naphtha, kerosene, diesel, lubricating oils, paraffin wax, tar
and petroleum coke are the products derived after distillation of crude oil
(WHO, 2000). Among all these products petrol, diesel, kerosene, LPG and
natural gas are the commonly used fuel in worldover.
Petrol is a one of the petroleum derived liquid mixture, it is produced
by the distillation, cracking and reforming of crude oil, primarily used
as fuel in internal combustion engines in automotive vehicles as well as
some aeroplane. Petrol is clear, pale brown or pink volatile liquid having
pungent odor at <1 ppm. Practically petrol is insoluble in water. Specific
gravity of Petrol varies between 0.71-0.77 g/cm3. Petrol contains over 500
hydrocarbons that may have between 3 to 12 carbons and gasoline boiling
range from 30°C to 220°C at atmospheric pressure (Diane et al., 2006).
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According to uses and region wise petrol is also known as Gasoline,
Gasolene, Mogas, Avgas and Benzin.
Petrol contains about 15% n-paraffins, 30 % iso-paraffins, 12%
cycloparaffins, 35% aromatics, 8% olefins, oxygenates and antiknock
agents. However, other commercial petrol mixtures may have different
compositions according to the manufacturers, with their own special
ingredients to provide additional benefits. A quality gasoline additive
package would include octane-enhancing additives, anti-oxidants, metal
deactivators, deposit modifiers, surfactants, freezing point depressants,
corrosion inhibitors and dyes (Gary and Handwerk, 2001; Speight, 2008).
The anti-knocking properties of gasoline are measured by octane
rating. The octane rating is not directly related to the amount of octane
contained in the gasoline but comparison with the mixture of 2,2,4trimethylpentane (iso-octane) and heptane which would have the same antiknocking capacity as the fuel under test: the percentage, by volume, of 2,2,4trimethylpentane in that mixture is the octane number of the fuel. For
example, petrol with the same knocking characteristics as a mixture of 90%
iso-octane and 10% heptane would have an octane rating of 90.
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In a number of countries tetraethyl lead has been used as a gasoline
additive and is emitted in small quantities from automobile exhaust.
Although it degrades quickly in the atmosphere, but it can be very hazardous
to gasoline sniffers. Tetraethyl lead is rapidly taken up by the nervous
system. Its toxicity depends on its activation in vivo to trialkyl forms. Upon
absorption into the body, it is converted to triethyl lead, diethyl lead and
inorganic lead (McKetta, 1992). Mandatory leaded petrol in India is
replaced by unleaded petrol which contains less than 0.013 g Pb/L.
Alternative gasoline fuel additives that may be used or have been
suggested to enhance the octane number when alkyl lead is completely
phased out include manganese oxide, methyl cyclopentadienyl manganese
tricarbonyl (MMT), tertiary butyl ether (TBE), ethanol, methanol, methyl
tertiary butyl ether (MTBE), benzene and toluene (Chris, 2007; Drew et al.,
2007; EPA, 1998) (Chris, 2007; Drew et al., 2006; EPA, 1998).
Ethanol and methanol may be added to commercial gasoline. Gasohol
is the name given to a mixture of gasoline and ethanol, which usually
contains 10% ethanol. In India, 5% ethanol is added within the common
petrol fuel. Where as, in European countries, they are discussing to allow
10% blending of ethanol. Most gasoline sold in Sweden has 5% ethanol
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added. In Brazil, 25% ethanol is blended with petrol. Brazil is the pioneer in
the field of biofuel production and implementation in the world, who use
85%, 90% and 95% ethanol as a biofuel. Where USA and Canada are using
40-60% ethanol as a biofuel.
Petrol is a mild skin, eye and respiratory tract irritant. Ingestion of
gasoline causes mild to severe irritation to the gastrointestinal mucosa,
chemical pneumonitis is often severe. Systemic effects of gasoline exposure
are mainly a result of CNS depression. Systemic effects can occur from all
routes of exposure. Acute exposure to low concentrations may produce
flushing of the face, staggering gait, slurred speech and mental confusion.
Higher concentrations may result in unconsciousness, coma and possible
death due to respiratory failure. Where as, in chronic exposure no ill effect
has been reported from by routine use of gasoline as a fuel (Collins et al.,
1991; Hallenbeck and Flowers, 1992; Lee et al., 1993).
The mandatory decrease of lead alkyls in petrol has led to an increase
in the aromatic hydrocarbon content of gasoline to maintain high octane
levels and/or antiknock properties. The specific density of petrol ranges from
0.71–0.77, but higher densities indicate a greater volume of aromatics. In
United States, gasoline typically contains less than 2% benzene by volume,
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but in India and other developing countries the benzene concentration may
be as high as 5% (Schultz et al., 2010)
Benzene
Benzene is the first member of a series of aromatic hydrocarbons
recovered from refinery streams during catalytic reformation and other
petroleum processes. In 1825, Michael Faraday first time discovered
Benzene (Faraday, 1825). But the structure of benzene was proposed by
Friedrich Kekulé in 1865, one of the significant contributor to organic
structural theory.
The chemical formula of benzene is C6H6, melting point is 5.5o C with
a density of 0.87 g/cm3 at 20oC and boiling point of Benzene is 80.1o C. It is
a clear, colorless, highly flammable liquid at room temperature. Benzene has
a sweet aromatic odor detectable at concentrations of 1.5 to 4.7 parts per
million
(ppm).
Common
synonyms
for
benzene
include
benzol,
cyclohexatriene, phenyl hydride and coal tar naphtha. It is slightly soluble in
water (1.8 g/litre at 25 oC) and miscible with most organic solvents (WHO,
2000).
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Trace amounts of benzene may result when carbon-rich materials
undergo incomplete combustion. It is produced in volcanoes and forest fires
and is also a component of cigarette smoke. Till World War II, most benzene
was produced as a byproduct of coke production in the steel industry. But,
increased
demand
for
benzene,
especially
from
the
growing plastics industry, necessitated the production of benzene from
petroleum. Today, most benzene comes from the petrochemical industry,
with only a small fraction being produced from coal. Three chemical
processes contribute equally to industrial benzene production: catalytic
reforming, toluene hydro-dealkylation and steam cracking.
In 19th and early-20th centuries, benzene was used as an after-shave
lotion because of its pleasant smell. Prior to the 1920s, benzene was
frequently used as an industrial solvent, especially for degreasing metal. As
its toxicity became obvious, benzene was supplemented by other solvents,
especially toluene (methyl benzene), which has similar physical properties
but is not as carcinogen. As a petrol additive, benzene increases the octane
rating and reduces knocking. Consequently, petrol often contained several
percentages of benzene before the 1950s, when tetraethyl lead replaced it as
the most widely-used antiknock additive. With the global phase-out of
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leaded petrol, benzene has made a comeback as a petrol additive (Verma et
al., 2003).
Today benzene is one of the world’s major commodity chemicals. Its
primary use approximately 85% of production is as an intermediate in the
production of other chemicals, predominantly styrene (for styrofoam and
other plastics), cumene (for various resins) and cyclohexane (for nylon and
other synthetic fibers). Benzene is an important raw material for the
manufacture of synthetic rubbers, lubricants, detergents, gums, napalm,
dyes, pharmaceuticals and agricultural chemicals.
Because of its lipophilic nature, benzene is an excellent solvent. Its
use in paints, thinners, inks, adhesives and rubbers, however, is decreasing
and now accounts for less than 2% of current benzene production. Benzene
was also an important component of many industrial cleaning and
degreasing formulations, but now has been replaced mostly by toluene,
acetone, chlorinated solvents, or mineral spirits. Although benzene is no
longer added in significant quantities to most commercial products, traces of
it may still be present as a contaminant (Haseeb, 2007).
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Route of exposure
Major route of benzene exposure is inhalation, about 99% of the
exposure in the general population, whereas intake by food and water is
minimal. Within the USA, the daily intake from ambient and indoor air has
been calculated to range between 180 and 1300 µg/day and the intake from
food and water up to about 1.4 µg/day (WHO, 2000). The average daily
intake for an adult in Canada was estimated to be 14 µg from ambient air,
140 µg from indoor air, 1.4 µg each from food and drinking water and 49 µg
from car-related activities, giving a total of 203 µg/day (Hughes et al.,
1994). Wallace et al., (1986) estimated the corresponding average intake in
the USA to be 320 µg/day. Cigarette smoking may add as much as 1800
µg/day and passive smoking 500 µg/day. Driving a car during the rush hour
may give a significant intake of air benzene.
Toxicokinetics
Absorption
Inhalation and ingestion are the two main pathways from where
benzene absorbs rapidly and extensively. Absorption through the skin is
rapid but not extensive, as most of it evaporates quickly (Nakai et al., 1997;
Wester, 2000). Human inhalation studies at 160–320 mg/m3 (50–100 ppm)
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for 4 hrs suggest approximately 50% absorption and 30% retention of the
inhaled dose, the rest being exhaled as unchanged benzene. An in vivo study
on human volunteers indicated that approximately 0.05% of a benzene dose
applied to the skin was absorbed, whereas in an in vitro study of human skin,
the absorption of benzene was consistently 0.2% after exposure to doses
ranging from 0.01 to 520 µl per square centimeter. Oral absorption has not
been studied in humans. In animals, at least 90% of benzene was absorbed
following oral ingestion of a dose of 340 to 500 milligrams per kilogram per
day (mg/kg/day). Benzene is distributed throughout the body with lipid-rich
and well perfused tissues, containing the highest levels. Benzene can cross
the placenta also.
Metabolism and elimination
Once absorbed, benzene is initially metabolized in the liver and later
in the bone marrow. Benzene metabolism by P-450 2E1system in the liver
involves oxidation, where it coverts into phenol as the major metabolite.
Further metabolic products are formed in liver and in bone marrow by the
enzymatic addition of hydroxyl groups to the benzene ring. Such metabolites
include hydroquinone, catechol and hydroxybenzene, which are further
conjugated and excreted in the urine. These hydroxylated metabolites can be
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further oxidized to their corresponding quinones or semiquinones. Benzene
oxide may also be metabolized via glutathione conjugation to form S-phenyl
mercapturic acid. Additionally, urinary excretion of small amounts of
muconic acid, a straight-chain dicarboxylic acid, indicates that the benzene
ring also is opened during metabolism. Trans, trans - muconic acid and
phenol are proven promising biomarkers of low level exposure of benzene
found from urine (Kim and Kim, 1996; Lee et al., 1993; Rappaport and
Kupper, 2004). The average half-time of benzene in humans is 28 hours,
whereas in rats and mice, metabolites are excreted in the urine within 40
hours of dosing by any route of administration (Medinsky et al., 1994).
Bone marrow is the main target organ of chronic benzene toxicity.
One or more benzene metabolite is suspected to be responsible for the
hematogenous toxicity, although the identity of the ultimate toxicant is
unknown. In the marrow, the metabolites may bind covalently to cellular
macromolecules (e.g., proteins, DNA and RNA), causing disruption of cell
growth and replication (Kolachana et al., 1993).
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Biomarkers of human exposure
At high exposure levels (above 31.9 mg/m3, 10 ppm) there is a
correlation between phenol excretion in urine and the level of exposure. At
lower concentrations, overall benzene exposure is reflected in the amount
exhaled in breath. Smokers have been found to exhale around 14 µg/m3 and
nonsmokers around 2 µg/m3 (Verma et al., 2003). The urinary excretion of
the specific benzene metabolite trans,trans-muconic acid has been found to
be enhanced in benzene-exposed workers and in smokers (Kim et al., 2002).
The excretion of 8-hydroxy-deoxyguanosine, formed as a result of oxidative
DNA damage, correlated with benzene exposure in petrol station attendants
(Lagorio et al., 1994). Possible adducts with benzene oxide such as Nphenylvaline or S-phenylcysteine in haemoglobin could not be detected in
benzene-exposed worker (Bader et al., 1994), although there was a linear
correlation of the latter adduct in albumin with benzene exposure (13-74
mg/m3) in female Chinese workers (Bechtold and Henderson, 1993).
Depending on the magnitude of the dose, persons who have ingested
benzene may experience these effects 30 to 60 minutes after benzene
ingestion. In one case report, an oral dose of 10 milliliters was reported to
produce staggering gait, vomiting, tachycardia, pneumonitis, drowsiness,
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delirium, seizures, coma and death. Early symptoms of chronic benzene
exposure are often nonspecific but show marked individual variability (Bois
and Paxman, 1992). By the time a physician is consulted, the bone marrow
may have been significantly affected. For example, conditions that first
bring the patient to medical attention are typically fever due to infection or
manifestations of thrombocytopenia, such as hemorrhagic diathesis with
bleeding from the gums, nose, skin, gastrointestinal tract, or elsewhere,
fatigue and anorexia.
Health effects
Physiologic Effects
Benzene exposure affects the CNS and hematopoietic system and may
affect the immune system. Death due to acute benzene exposure has been
attributed to asphyxiation (suffocation), respiratory arrest, CNS depression,
or cardiac dysrhythmia. Pathologic findings in fatal cases have included
respiratory tract inflammation, lung hemorrhages, kidney congestion and
cerebral edema (Kanada et al., 1994; Ungvary and Donath, 1984).
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Hematologic Effects
Benzene can cause dangerous hematologic toxicity such as anemia,
leucopenia, thrombocytopenia, or pancytopenia after chronic exposure.
These effects are believed to be caused by the metabolites of benzene, which
most likely damage the DNA of the pluripotential stem cells. All of the
blood’s components [i.e., erythrocytes, leukocytes and thrombocytes
(platelets)] may be affected to varying degrees. The accelerated destruction
or reduction in the number of all three major types of blood cells is termed
pancytopenia. Potentially fatal infections can develop if granulocytopenia is
present and hemorrhage can occur as a result of thrombocytopenia.
Cytogenetic abnormalities of bone marrow cells and circulating lymphocytes
have been observed in workers exposed to benzene - abnormalities not
unlike those observed after exposure to ionizing radiation. Myelodysplastic
effects also can be seen in the bone marrow of persons chronically exposed
to benzene (Chertkov et al., 1992; Infante et al., 1977).
Aplastic anemia is caused by bone marrow failure, resulting in
hypoplasia with an inadequate number of hematopoietic stem cells. Severe
aplastic anemia typically has a poor prognosis and can progress to leukemia,
whereas pancytopenia may be reversible. Benzene-induced aplastic anemia
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is generally caused by chronic exposure at relatively high doses. Fatal
aplastic anemia following benzene exposure was first reported in workers in
the nineteenth century (Kissling and Speck, 1972).
Environmental Protection Agency [EPA] and the International
Agency for Research on Cancer classify benzene as a confirmed human
carcinogen. EPA estimates that a lifetime exposure to 4 ppb benzene in air
will result in, at most, 1 additional case of leukemia in 10,000 people
exposed. EPA has also estimated that lifetime exposure to a benzene
concentration of 100 ppb in drinking water would correspond to, at most, 1
additional cancer case in 10,000 people exposed. Studies of benzeneexposed workers in several industries e.g., sheet-rubber manufacturing, shoe
manufacturing and refinery industries, have demonstrated significantly
elevated risk of leukemia predominantly Acute Myelogenous Leukemia
(AML), but also Erythroleukemia and Acute Myelomonocytic Leukemia.
The dormant period for benzene-induced leukemia is typically 5 to 15 years
after first exposure. Patients with benzene-induced aplastic anemia progress
to a preleukemic phase and develop Acute Myelogenous Leukemia.
However, a person exposed to benzene may develop leukemia without
having aplastic anemia.
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Standards and regulation
Occupational safety and health administration (OSHA) suggested
permissible exposure limit (PEL) of benzene at workplace is 1 ppm (1 ppm
= 3.19 mg/m3) for normal 8 hour/day and 5 ppm for short term exposure
limit (15 minutes TWA - time weighed average). The National Primary
Drinking Water Regulations promulgated by EPA in 1987 set a maximum
contaminant level for benzene of 0.005 ppm (5 ppb). This regulation is
based on preventing benzene leukemogenesis. The maximum contaminant
level would allow an adequate margin of safety for the prevention of adverse
effects, is zero benzene concentration in drinking water (OSHA, 2005).
Studies indicate that the risk of leukemia is associated with
occupational low level exposures to benzene (<1 ppm). A large number of
industrial workers from petroleum, rubber, paint, shoe making, printing,
solvent and other chemical industries are occupationally exposed to benzene
(Pekari et al., 1992).
Aksoy (1987) and Hayes et al., (2001) concluded that occupational
exposure to benzene has mainly been associated with increased incidences
of Acute Myeloid Leukemias, but also with chronic myeloid and acute
lymphoid leukemia, multiple myeloma
and non-Hodgkin's lymphomas
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(Goldstein and Cody, 2000; Miller et al., 1994; Wong and Raabe, 2000).
Kesavachandran et al., (2006) observed some lung function abnormaliles.
In addition, Smith and Rothman, (2000) and Zhang et al.(1996, 2002,
2005) reported that occupational benzene exposure may increase aneusomy
and long arm deletion of chromosome no 5 and 7. Epidemiological studies
also showed that there is a clear relationship between the increase in
micronuclei (MN) frequency and exposure to benzene and benzene
metabolites (Tompa et al., 1994; Turkel and Egeli, 1994; Yager et al.,
1990).
Few studies indicate that sister chromatid exchange (SCE) frequency
increases due to benzene exposure (Major et al., 1994) where as some
studies indicate that benzene doesn’t increase sister chromatid exchange
frequency but its metabolites like, phenol, catechol and hydroquinone
responsible to increase SCE (Zhong, 1982). Seiji et al. (1990) did not find
any significant change in SCE frequency between cigarette smokers and non
smokers. Bukvik et al., (1998) revealed increased sister chrometid exchange
and micronuclei frequency in lymphocyte of gasoline station attendants.
There are reported increased SCE frequencies and chromosomal
aberrations in petrol pump workers (Bukvic et al., 1998; Celik et al., 2005;
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Yadav and Seth, 2001). Celik et al., (2003) shows increased micronuclei
frequency in exfoliated buccal cells of gasoline station attendants. Tunca
and Egeli (1996) revealed that shoe workers having higher chromosomal
and chromatid aberrations than control individuals.
Verma et al., (2003) observed increased phenol level in traffic police
men. In other studies, Rothman et al., (1998) observed urinary catechol and
hydroquinone increase with benzene exposure, where as Wiwanitkit et al.,
(2007) found increased urinary phenol and trans, trans - muconic acid level
from mechanics working in garages.
In the case of petrol pump attendants they are occupationally exposed
to chronic and low dose of benzene exposure during whole day shift by
inhaling volatile petrol fume containing benzene. In addition, cigarette
smoke contain small amount of benzene, means who are cigarette smokers,
getting more benzene than other non smoker petrol pump exposed workers.
Our purpose of study is to investigate cytogenetic effect of benzene from
lymphocytes of petrol pump workers by using sister chromatid exchanges,
micronuclei
and
chromosomal
aberrations.
Furthermore
we
have
investigated urine of petrol pump attendants to determine phenol and trans,
trans – muconic (t,t-MA) acid to ensure benzene exposure. Because, t,t-MA
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is proven as a very good biomarker of chronic benzene exposure. To
compare chromosomal aberrations due to benzene exposure, mitomycin-C is
used as a positive control. We also took hematological parameters, like total
WBC, RBC and Platelet count, Hb, PCV and differential count.
The present work comprised of total 100 male volunteer subjects,
these subjects further classified into two groups; Group A and Group B.
Group A and Group B further classified into two sub groups A1, A2, B1and
B2 respectively, according to their smoking addiction. Each group consists of
25 individuals.
Group A1: Individuals who are non smoker and occupationally not
exposed to benzene.
Group A2: Individuals who are smoker and occupationally not
exposed to benzene.
Group B1: Petrol pump attendants who are non smokers.
Group B2: Petrol pump attendants who are smokers.
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