Mutation Research 700 (2010) 67–70
Contents lists available at ScienceDirect
Mutation Research/Genetic Toxicology and
Environmental Mutagenesis
journal homepage: www.elsevier.com/locate/gentox
Community address: www.elsevier.com/locate/mutres
Ultraviolet radiation-induced genotoxic effects in the broad-snouted caiman,
Caiman latirostris
L.G. Schaumburg a , G.L. Poletta a,b,c,∗ , A. Imhof a , P.A. Siroski a,d
a
“Proyecto Yacaré” – Laboratorio de Zoología Aplicada: Anexo Vertebrados (FHUC - UNL/MASPyMA), A. del Valle 8700, Santa Fe, Argentina
Cátedra de Toxicología y Bioquímica Legal, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, Santa Fe, Argentina
Grupo de Investigación en Biología Evolutiva, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires – CONICET, Buenos Aires, Argentina
d
Secretaria de Estado de Medio Ambiente y Desarrollo Sustentable de la Provincia de Santa Fe, Santa Fe, Argentina
b
c
a r t i c l e
i n f o
Article history:
Received 4 February 2010
Received in revised form 27 April 2010
Accepted 4 May 2010
Available online 11 May 2010
Keywords:
UV radiation
Crocodilians
Caiman latirostris
Micronucleus test
Genotoxicity
a b s t r a c t
Ultraviolet radiation (UVR) has many positive effects, but overexposure of organisms can generate alterations in DNA, either directly or indirectly, inducing mutagenic and cytotoxic lesions. The aim of this
study was to evaluate the genotoxic effects of UVA–B/visible light in the broad-snouted caiman (Caiman
latirostris), using the micronucleus (MN) test as a biomarker.
Seventy two juvenile caimans, approximately 5 months old, were maintained for during 3 months
under total darkness, or under 8 or 16 h of daily exposure to artificial UV/visible light. MN test was applied
before and after the experiment and the difference in MN frequencies was determined. Our results indicate significant increases in MN frequency with all treatments, compared to the basal (before experiment)
values. Animals exposed to UV radiation showed a greater increase in MN frequency, compared to the
animals exposed to total darkness (TD) treatment.
These results provide information about the possible harmful effects generated by sub-chronic exposure to UVR in zoos, reptile hobbyist and breeding programs, as well as the deleterious consequences of
increased UV environmental impact on wild species such as the broad-snouted caiman.
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
Sunlight is a key environmental factor in almost all ecosystems,
and favorable ecological effects of visible and infrared wavelengths have been known for a long time [1]. Ultraviolet (UV)
light forms a part of the solar emission spectrum, with a wavelength range of 200–400 nm, corresponding to 7.2% of the total light
energy [2]. The UV range can be divided into three wavelengths
bands: long wavelength (UVA), 315–400 nm (5.1%); medium wavelength (UVB), 280–315 nm (1.3%); and short wavelength (UVC),
200–280 nm (0.8%) [1–3]. Of these three bands, UVC does not reach
the biosphere, because it is strongly absorbed by oxygen, ozone,
and other gases [1,3,4]. In contrast, UVA and UVB radiation (UVR)
reach the biosphere, although the latter is predominantly absorbed
by atmospheric ozone [1].
In living organisms, UVA is absorbed by many proteins and chromophores in living cells, and the radiation can stimulate several
∗ Corresponding author at: “Proyecto Yacaré” – Laboratorio de Zoología Aplicada:
Anexo Vertebrados (FHUC - UNL/MASPyMA), A. del Valle 8700, Santa Fe, Argentina.
Tel.: +54 342 4579256; fax: +54 342 4579256.
E-mail addresses: [email protected], [email protected]
(G.L. Poletta).
1383-5718/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.mrgentox.2010.05.002
physiological processes. UVA is also detected by photoreceptors
in the eyes of both vertebrates and invertebrates [1]. In most animals, including humans, UVB radiation is needed for vitamin D3
synthesis [5], which is fundamental for the regulation of calcium
metabolism [6,7] as well as for some immune functions [8]. In spite
of the positive effects of UVR on organisms, as a result of ozone layer
reduction in some areas of the world, there has been an increase in
UVR incidence on ecosystems, representing a potential hazard to
living organisms [9,3].
The influence of natural or artificial light on animal growth in
caiman captive breeding systems and management programs has
been little studied [10]. The change in natural sunlight conditions in
these systems can produce alterations in production of vitamin D3 ,
so additional use of full spectrum and low intensity lamps has been
implemented for normal development and function under these
conditions [11,12].
Excessive UVR exposure may induce adverse biologic effects due
to photochemical absorption by important biomolecules, such as
nucleic acids and proteins [13,14]. Among these adverse effects,
DNA damage induced by UVA/UVB exposure occurs mainly by
direct excitation of the nitrogenous bases, leading to mutations
and chromosome fragmentation [15,16]. The alterations most frequently induced are cyclobutane pyrimidine dimers (CPDs) and
pyrimidine (6–4) pyrimidone photoproducts (6–4PPs) [17–19].
68
L.G. Schaumburg et al. / Mutation Research 700 (2010) 67–70
CPDs are formed between the 5 and 6 bonds of two adjacent pyrimidine bases. The 6–4PPs are characterized by a stable bond between
positions 6 and 4 of two neighbouring pyrimidines. However, they
occur at a rate substantially lower than CPDs [20,21]. Photoproducts
generated in lesser amounts include purine dimers and pyrimidine
monoadducts [13].
UVR can also induce alterations in DNA indirectly, through
photosensitization reactions, promoting free radical and reactive
oxygen species (ROS) formation. ROS may oxidize the bases, leading to DNA breaks [22,16,23]. UV photoproducts can block DNA
replication [24], alter gene expression by preventing transcription,
and cause mutagenesis or cell death, depending on the altered DNA
sequence/s [3,25].
The MN test is a biomarker of genotoxicity that allows the detection of effects produced by agents which cause both chromosome
fragmentation (clastogenesis) and/or malfunctions of the spindle
apparatus or the centromere–kinetochore complex, affecting chromosome segregation (aneugenesis) [26–29]. The test has been used
to evaluate UVR-induced genotoxicity in many organisms and cell
lines [30,15,31,9,32].
Only one previous report describes application of the MN test
in crocodilian species and other reptiles [33]. Recently, however,
the MN test has been validated as a biomarker of genotoxicity in
erythrocytes of Caiman latirostris [34]. C. latirostris is one of the
two crocodilian species found in Argentina. Its distribution covers
a wide area in the northeastern region of the country [35,36]. In
Santa Fe, C. latirostris occurs in a wide range of aquatic habitats
throughout the Paraná and Salado river basins [37].
This species has great ecological importance for an extensive
region of the country. However, no studies have yet been carried
out on the effects of UV radiation on this animal. The aim of this
study was to use the MN test to evaluate the genotoxic effects of
UVA and UVB radiation in juvenile C. latirostris.
2. Materials and methods
2.1. Animals
Seventy two juvenile broad-snouted caiman, approximately 5 months old,
hatched from three nests harvested in the wild and artificially incubated as part of
Proyecto Yacaré (PY) ranching program [36], were used. They had been maintained
in pools at PY commercial husbandry facilities since hatching.
2.2. Experimental design and treatments
The experiment lasted 3 months. Animals were maintained in plastic chambers
(96 cm long, 41 cm wide and 42 cm high, surface = 0.39 m2 ), tilted to give 70% dry
and 30% water surface areas, with a maximum water depth of approximately 15 cm
and fully covered with a dark plastic (nylon), to avoid the penetration of external
light. Inside the chambers, temperature was maintained at 31 ± 2 ◦ C, monitored with
Hobo Temperature recorders (Onset Computer Corp., Pocasset, MA).
Each animal was individually marked with foot webbing tags (Monel National
Band and Tag Co., Newport, KY, 1005-1), and four animals from each nest were
randomly assigned to each of two replica (12 caimans per replica) of the different
treatment groups, giving a total of 24 caimans per group. Experimental treatments
were: total darkness (TD), eight hours (T8 ) and sixteen hours (T16 ) of artificial
UV/visible light exposure, each with a corresponding replica.
During the experiment, animals were maintained under identical conditions
(temperature, feed and cleaning regimes [38]). They were fed ad libitum three times
per week with a mixture of 50% minced chicken head and 50% dry pellets. The
cleaning of pens was also carried out three times per week (alternating with feeding,
in order to ensure sanitary conditions).
All animals were measured for total length (TL) and snout-vent length (SVL),
with a precision of 0.5 cm, and weighed with a precision of 0.1 g (Electronic compact
scale, TH 5000) at the beginning and end of the experiment.
2.3. UV exposure
UV exposure was carried out with an 18 W visible–UV light lamp with 30%
UVA and 5% UVB, used in reptilian husbandry (Sylvania Reptistar® ). The lamp was
set 0.45 m above the base of the plastic chambers and provided an irradiance of
45.73 W/m2 (irradiance = lamp power/plastic chamber surface).
Fig. 1. Increase in MN frequency (final-basal MN values) in different treatments
(mean ± S.D.) TD: total darkness, T8 : 8 h UV/visible light exposure, T16 : 16 h
UV/visible light exposure. Number of animals per treatment: 24. *Significantly different respect to TD treatment (Mann–Whitney U-test).
2.4. Blood collection
At the beginning and end of the experiment, peripheral blood samples (0.5 ml)
were obtained from each animal via the spinal vein [39,40], with 5 ml heparinized
syringes and 25 ga needle.
2.5. Micronucleus test
The MN assay was performed according to Schmid [26] with modifications introduced by Poletta et al. [34] to be applied in C. latirostris erythrocytes. Blood samples
collected before the experiment were used to determine basal MN frequency for all
animals, as a reference prior to exposure, while blood collected after the experiment
was used for determination of post-exposure MN frequency. Two blood smears were
made for each animal on clean glass slides, fixed with methanol for 10 min, and then
stained with Giemsa for 10 min. Giemsa solution was centrifuged and filtered before
staining to reduce precipitations that could be confuse with MN and interfere with
analysis. Slides were coded for ‘blind’ analysis and then examined under an optical
microscope at 1000×. For each individual, 1000 erythrocytes were analyzed and the
frequency of micronucleated cells among them was recorded.
2.6. Statistical analysis
Statistical analysis was performed using the software SPSS 14.0 for Windows
[41]. Variables were tested for normality with the Kolmogorov–Smirnov test and
homogeneity of variances between groups was verified by the Levene test. Basal
MN values were analyzed by one-way ANOVA with treatment and nest of origin
as grouping variables and the frequency of MN as response variable. The difference
in MN frequency (final − initial values) was analyzed by non-parametric tests. We
used the Kruskal–Wallis, followed by the Mann–Whitney test for the comparison
of MN frequencies between experimental treatments. We applied the Bonferroni
correction according to the number of analyses by pairs carried out, so that a p value
<0.016 was considered significant.
3. Results
Mean basal MN frequency (before exposure) determined from
all animals was 0.40 ± 0.72. A single basal MN frequency is used
as the mean for all animals (N = 72), because no differences were
observed in MN values either between treatments to which animals were assigned or between clutches (p = 0.832 and 0.179,
respectively), confirming the random distribution of animals at the
beginning of the experiment.
We found no differences between replicas after exposure to any
treatments (p > 0.05), so all of the results are presented as mean
values ± standard deviation (S.D.) per experimental group. MN frequency after UV exposure was significantly higher in all treatments
compared to basal values (p < 0.001), but there were differences in
the increase, depending on the treatment. Animals in the T8 and T16
groups presented a significantly higher increment in MN frequency
(final − initial values: 7.21 ± 1.58 and 7.50 ± 1.07, respectively)
than the TD groups (1.75 ± 0.38; p < 0.001). However, no difference
was found between the T8 and T16 treatments (p = 0.305) (Fig. 1).
A clear “clutch effect” was observed in the incidence of MN frequency after exposure to different treatments (p < 0.001), but there
was no interaction between nest and treatment (p = 0.342). Similarly, the increase in MN frequency was not influenced by basal
values (p = 0.270).
L.G. Schaumburg et al. / Mutation Research 700 (2010) 67–70
4. Discussion
The increased incidence of UVR is an environmental problem
affecting many regions in the world, with effects on the individual,
population, community, and ecosystem [42]. Many studies have
been conducted on the mutagenic effects of solar radiation on
humans, demonstrating deep cell damage, mainly in the skin, after
chronic exposure. However, relatively few studies have evaluated
the effects of UVR on wildlife.
Some species and some individuals have greater capacity to
tolerate UVR, due to more effective protective and repair mechanisms [23]. There are two major mechanisms for repair of UV
induced damage: nucleotide excision repair (NER), also known
as “dark repair”, a general DNA repair mechanism that can act
on both CPDs and 6–4PPs, in the absence of light; and photoenzymatic repair (PER) or “light repair”, which involves the direct
monomerization of CPDs catalyzed by the enzyme photolyase, in
the presence of suitable temperatures and light, especially UVA
and short-wavelength visible light [16,23,43]. PER is one of the
most ancient DNA repair mechanisms, unlike the other pathway,
but it has only been observed in a few organisms, such as certain marine plankton, bacteria, fish, and amphibians [43]. Oxidative
DNA damage is repaired primarily by the base excision repair
(BER) pathway, which acts on small DNA lesions such as oxidized bases, abasic sites, and DNA single-strand breaks [43]. When
protective and repair mechanisms are overwhelmed as a result
of UVR exposure, DNA photoproducts can accumulate, leading to
seriously affected populations. In ectothermic organisms, such as
the broad-snouted caiman, low temperatures in natural conditions can reduce PER. Therefore, these animals are more likely to
suffer from an imbalance between damage and repair processes
[13].
The results of our study revealed that the MN frequency in the
erythrocytes of C. latirostris after in vivo exposure increased in all
treatments (TD, T8 and T16 ), in comparison to the basal values. The
increase observed in TD treatment was significantly less than in
either group exposed to UVR, and indicates a negative effect due to
total lack of natural light. In contrast, the increase observed in both
T8 and T16 indicated a genotoxic effect of UVR on erythrocytes of
exposed animals.
Previous studies have demonstrated genotoxic effect of UVR
in vitro and in vivo in various cell populations and organisms.
Wang and Wang [9] studied the in vitro effect in Tradescantia after
exposure to four treatments of UVB, showing an increase in MN
frequency with UV dose. They also noted that in the presence of
higher solar radiation background, the damage generated was even
higher, showing an additive effect. Botta et al. [44] demonstrated
genotoxic effects induced by UVA/visible light, by use of the comet
assay (CA) in normal human keratinocytes and the in vitro MN test
in CHO cells. Both assays showed a significant increase in DNA
damage and a clearly dose-dependent effect. Lankoff et al. [45]
determined the effects of UVR on NER in the CHO cell line, using
the MN test to evaluate DNA damage and the CA to determine
the kinetics of NER. There was a significant negative correlation
between results observed with the two tests, indicating that those
cells with a decreased in the ability of NER presented higher MN
frequencies, corresponding to a high level of DNA damage induced
by UVR. On the other hand, in vivo studies conducted to evaluate UVA/UVB exposure in two species of neotropical fish revealed
genotoxicity in erythrocytes as measured by the CA but not the MN
test [46]. It was also found that DNA damage was repaired after
12–48 h without UV exposure. The authors indicated that these
results showed that the damage generated by acute UV exposure
(120 min) was repairable. For that reason, the CA, which detects
lesions that can be repaired such as breaks and alkali-labile sites,
showed positive results, whereas the MN test, which detects non-
69
repairable damages, such as clastogenic and aneugenic lesions,
showed negative results [46]. In contrast, in our study, the MN test
indicated DNA damage in both exposed groups. This could reflect
the sub-chronic (3 months) UV exposure condition, since the MN
test is suitable for detection of chronic exposure effects to genotoxic
agents, while the CA is more useful for detecting DNA damage and
repair induced as a result of recent exposure [46]. Sastre et al. [47]
reported genotoxicity in the marine unicellular algae Rhodomonas
sp. after exposure to 12 h visible + UVA + UVB radiation, compared
to a control exposed only to visible light. They used the CA modified for the detection of pyrimidine dimers with T4 endonuclease
V. In most of these studies, treatments consisted of different doses
of UVR, while in our study, the difference in the UV treatments was
the length of exposure to the same irradiance (45.73 W/m2 with
30% UVA and 5% UVB). However, although T16 received twice the
length of UVR exposure, compared to T8 , the increment of MN frequency observed was similar. This indicates that, in the present
study, the UVR effect was already maximal at the lower exposure
level.
In order to compare the UVR exposure used in this study with
natural environmental exposure to sunlight, it is important to clarify that this study was done in Santa Fe province, Argentina, where
the solar irradiance average during the time of year when the
study was conducted is approximately 150 W/m2 [48]. Although
sunlight irradiance is approximately three times greater than that
of the lamp, animals in natural environments can regulate their
solar exposure, and they typically spend only a few hours per day
“sunbathing”. Consequently, little or no damage might be expected
in wild populations under natural conditions. Nevertheless, the
increased incidence of UVR remains of concern with regard to
wildlife species.
One of the principal factors introducing variability in studies on
crocodilians is the “clutch effect” [49–51]. In our study, specimens
from different clutches showed different responses to treatment.
However, previous studies in this species reported no difference
between clutches in the spontaneous frequency of MN in animals
of different age or sex [34], nor in the increased frequency of MN
in neonates after in ovo exposure to an herbicide formulation [52].
These results could be explained by polymorphisms in enzymes
that repair UVR but not chemical damage. Another possibility is
that there are susceptibilities related to hereditary and/or acquired
components which are expressed in the animals of some clutches
but not in others [53].
The results of the present study – the first in which genotoxic
effects of UVR have been studied in vivo in a reptile – provide information about possible harmful effects resulting from sub-chronic
exposure to UVR in breeding programs and the deleterious consequences of increased UV environmental impact on wild reptilian
species.
5. Conclusions
UVR exposure induces a significant increment in the MN frequency in juvenile C. latirostris, demonstrating the genotoxic effect
of this agent. Despite differences in the length of UV exposure, both
treatments caused the same level of damage. Differences in the
effects of treatments observed in animals coming from different
clutches could be a result of genetic or nutritional variations in DNA
protection and repair mechanisms.
Conflict of interest
There is no competing interest.
70
L.G. Schaumburg et al. / Mutation Research 700 (2010) 67–70
Acknowledgements
This study was supported by Proyecto Yacaré and Yacarés
Santafesinos (MUPCN) and Universidad Nacional del Litoral (UNLSanta Fe, Argentina). The authors are grateful to other members of
Proyecto Yacaré for their collaboration in this study, to the staff of
the laboratory “L3”, Facultad de Humanidades y Ciencias, Universidad Nacional del Litoral (FHUC-UNL), and to Prof. Ricardo Carreri
(FHUC-UNL) for their help in UVR issues.
References
[1] D.P. Nigel, D. Gwynn-Jones, Ecological roles of solar UV radiation: towards an
integrated approach, Trends Ecol. Evol. 18 (2003) 48–55.
[2] F. Tena, J.A. Martínez-Lozano, M.P. Utrillas, La radiación solar ultravioleta y
prevención del eritema, Rev. Esp. Fís. 12 (1998) 18–24.
[3] A. Blaustein, N. Sengsavanh, Ultraviolet Radiation, Encyclopedia of Biodiversity,
Academic Press, 2001.
[4] R.A.C. Keulers, M.M. Corrie Van Teylingen, A.D. Tates, Effects of deoxyribonucleosides and cell-stimulation on frequencies of ultraviolet-B-induced
micronuclei in human peripheral blood lymphocytes, Mutat. Res. 459 (2000)
115–122.
[5] E.N. Carman, G.W. Ferguson, W.H. Gehrmann, T.C. Chen, M.F. Holick, Photobiosynthetic opportunity and ability for UV-B generated vitamin D synthesis
in free-living house geckos (Hemidactylus turcicus) and Texas spiny lizards
(Sceloporus olivaceous), Copeia 2000 (2000) 245–250.
[6] A. Marsden, Sunlight and Reptile UVB Tubes, The value of UVB exposure
http://www.anapsid.org/uvbanne.html, 1999.
[7] K.M Dixon, R.S. Mason, Vitamin D, Int. J. Biochem. Cell Biol. 41 (2009) 982–985.
[8] H. Brames, Aspects of light and reptilian immunity, Iguana 14 (2007) 19–23.
[9] S. Wang, X. Wang, The Tradescantia-micronucleus test on the genotoxicity of
UV-B radiation, Mutat. Res. 426 (1999) 151–153.
[10] G. Principe, Influencia del fotoperíodo en el crecimiento de Caiman latirostris
(Daudin. 1802) en condiciones experimentales, Tesina de Licenciatura en Biodiversidad, Facultad de Humanidades y Ciencias, U.N.L., Santa Fe, 2007.
[11] F.L. Frye, Reptile Care: An Atlas of Diseases and Treatments, TFH Publications
Inc., Neptune City, USA, 1991.
[12] H. Seufer, Keeping and Breeding Geckos, TFH Publications Inc., Neptune City,
USA, 1991.
[13] M.M. Caldwell, L.O. Bjhn, J.F. Bornman, S.D. Flint, G. Kulandaivelu, A.H. Teramura, M. Tevini, Effects of increased solar ultraviolet radiation on terrestrial
ecosystems, J. Photochem. Photobiol. B 46 (1998) 40–52.
[14] B.L. Diffey, What is light? Photoderm. Photoinmunol. Photomed. 18 (2002)
68–74.
[15] C. Keshava, T. Ong, J. Nath, Comparative studies on radiation-induced micronuclei and chromosomal aberrations in V79 cells, Mutat. Res. 328 (1995) 63–71.
[16] J. Cadet, E. Sage, T. Douki, Ultraviolet radiation-mediated damage to cellular
DNA, Mutat. Res. 571 (2005) 3–17.
[17] G.R. Hoffmann, Genetic toxicology, in: M.O. Amdur, J. Doull, C.D. Klassen (Eds.),
Casarett and Doull’s Toxicology, The Basic Science of Poisons, Pergamon Press
Inc., New York, 1991, pp. 280–296.
[18] S. Courdavault, C. Baudouin, M. Charveron, B. Canguilhem, A. Favier, J. Cadet, T.
Douki, Repair of the three main types of bipyrimidine DNA photoproducts in
human keratinocytes exposed to UVB and UVA radiations, DNA Repair 4 (2005)
836–844.
[19] G.P. Pfeifer, Y.H. You, A. Besaratinia, Mutations induced by ultraviolet light,
Mutat. Res. 571 (2005) 19–31.
[20] D.L. Mitchell, R.S. Nairn, The biology of the (6–4) photoproduct, Photochem.
Photobiol. 49 (1989) 805–819.
[21] J.H. Yoon, C.S. Lee, T. O’Connor, A. Yasui, G.P. Pfeifer, The DNA damage spectrum
produced by simulated sunlight, J. Mol. Biol. 299 (2000) 681–693.
[22] K. Bender, C. Blattner, A. Knebel, M. Lordanov, P. Herrlich, H.J. Rahmsdorf, UVinduced signal transduction, J. Photochem. Photobiol. B 37 (1997) 1–17.
[23] D.P. Häder, R.P. Sinha, Solar ultraviolet radiation-induced DNA damage in
aquatic organisms: potential environmental impact, Mutat. Res. 571 (2005)
221–233.
[24] B.A. Britt, Repair of DNA damage induced by ultraviolet radiation, Plant Physiol.
108 (1995) 891–896.
[25] A.A. Vink, L. Roza, Biological consequences of cyclobutane pyrimidine dimmers,
J. Photochem. Photobiol. B 65 (2001) 101–104.
[26] W. Schmid, The micronucleus test, Mutat. Res. 31 (1975) 9–15.
[27] B. Miller, F. Pötter-Locher, A. Seelbach, H. Stopper, D. Utesch, S. Madle, Evaluation of the in vitro micronucleus test as an alternative to the in vitro
chromosomal aberration assay: position of the GUM working group on the
in vitro micronucleus test, Mutat. Res. 410 (1998) 81–116.
[28] M.A. Campana, A.M. Panzeri, V.J. Moreno, F.N. Dulout, Micronuclei induction in
Rana catesbeiana tadpoles by the pyrethroid insecticide lambda-cyhalothrin,
Genet. Mol. Biol. 26 (2003) 99–103.
[29] S.M. Malladi, H.N. Bhilwade, M.Z. Khan, R.C. Chaubey, Gamma ray induced
genetic changes in different organs of chick embryo using peripheral blood
micronucleus test and comet assay, Mutat. Res. 630 (2007) 20–27.
[30] I.B. Bahari, F.M. Noor, N.M. Daud, Micronucleated erythrocytes as an assay to
assess actions by physical and chemical genotoxic agents in Clarias gariepinus,
Mutat. Res. 313 (1994) 1–5.
[31] E.M. Weller, T.R. Kinder, M. Köfferlein, W. Burkart, M. Nüsse, UV-B-induced
cell cycle perturbations, micronucleus induction, and modulation by caffeine
in human keratinocytes, Int. J. Radiat. Biol. 69 (1996) 371–384.
[32] I. Udroiu, The micronucleus test in piscine erythrocytes, Aquat. Toxicol. 79
(2006) 201–204.
[33] G. Zúñiga-González, O. Torres-Bugarín, A. Zamora-Pérez, B.C. Gómez-Meda,
M.L. Ramos-Ibarra, S. Martínez-González, A. González-Rodríguez, J. LunaAguirre, A. Ramos-Mora, D. Ontiveros-Lira, M.P. Gallegos-Arreola, Differences
in the number of micronucleated erythrocytes among young and adult animals including humans. Spontaneous micronuclei in 43 species, Mutat. Res.
494 (2001) 161–167.
[34] G.L. Poletta, A. Larriera, E. Kleinsorge, M.D. Mudry, Caiman latirostris (broadsnouted caiman) as a sentinel organism for genotoxic monitoring: basal values
determination of micronucleus and comet assay, Mutat. Res. 650 (2008)
202–209.
[35] A. Yanosky, Histoire naturelle du Caiman à museau large (Caiman latirostris),
un alligatoriné mal connu, Rev, Fr. Aquariol. 17 (1) (1990) 19–31.
[36] A.L. Larriera, A. Imhof, P. Siroski, Estado actual de los programas de conservación y manejo del género Caiman en Argentina, in: J. Castroviejo, J.
Ayarzaguena, A. Velasco (Eds.), Contribución al conocimiento del Género
Caiman de Sudamérica, Public. Asoc. Amigos de Doña Ana 18, Sevilla, España,
2008, pp. 294–300.
[37] A. Larriera, Áreas de nidificación y momento óptimo de cosecha de huevos
de Caiman latirostris en Santa Fe, Argentina, in: A. Larriera, L.M. Verdade
(Eds.), La conservación y el manejo de Caimanes y Cocodrilos de América
Latina, Fundación Banco Bica, Santo Tomé, Santa Fe, Argentina, 1995, pp. 221–
223.
[38] A. Larriera, A. Imhof, Proyecto Yacaré: Cosecha de huevos para cría en granjas
del género Caiman en la Argentina, in: M. Bolkovic, D. Ramadori (Eds.), Manejo
de Fauna Silvestre en la Argentina. Programas de Uso Sustentable, Dirección de
Fauna Silvestre, Secretaría de Ambiente y Desarrollo Sustentable, Buenos Aires,
2006, pp. 51–64.
[39] K.C. Zippel, H.B. Lillywhite, C.R.J. Mladinich, Anatomy of the crocodilian spinal
vein, J. Morphol. 258 (2003) 327–335.
[40] S. Tourn, A. Imhof, A. Costa, C. Von Finck, A. Larriera, Colecta de sangre y procesamiento de muestras en Caiman latirostris, in: Memorias del IV Workshop
sobre Conservación y Manejo del Yacaré Overo (Caiman latirostris), in: “La
Región” – Fundación Banco Bica – Santo Tomé, Santa Fe, Argentina, 1994, pp.
25–30.
[41] SPSS for Windows, 2005, Version 14.0, SPSS Inc. Chicago, USA, Available at:
http://www.spss.com/.
[42] A. Hamman, F.R. Momo, A. Duhour, L. Falco, M.C. Sagario, M.E. Cuadrado, Effect
of UV radiation on Eisenia fetida populations, Pedobiology 47 (2003) 842–845.
[43] Q. Dong, K. Svoboda, R.T. Terrence, W.T. Monroe, Photobiological effects of UVA
and UVB light in zebrafish embryos: evidence for a competent photorepair
system, J. Photochem. Photobiol. B 88 (2007) 137–146.
[44] C. Botta, C. Di Giorgio, A.S. Sabatier, M. De Méo, Genotoxicity of visible light
(400-800 nm) and photoprotection assessment of action, l-ergothioneine and
mannitol and four sunscreens, J. Photochem. Photobiol. B 91 (2008) 24–34.
[45] A. Lankoff, J. Sochacki, L. Spoof, J. Meriluoto, A. Wojcik, A. Wegierek, L. Verschaeve, Nucleotide excision repair impairment by nodularin in CHO cell lines
due to ERCC1/XPF inactivation, Toxicol. Lett. 179 (2008) 101–107.
[46] A.A. Groff, J. da Silva, E.A. Nunes, M. Ianistcki, T.N. Guecheva, A. Miranda de
Oliveira, C.P. Feitosa de Oliveira, A.L. Val, J.A.P. Henriques, UVA/UVB-induced
genotoxicity and lesion repair in Colossoma macropomum and Arapaima gigas
Amazonian fish, J. Photochem. Photobiol. B 99 (2010) 93–99.
[47] M.P. Sastre, M. Vernet, S. Steinert, Single-cell gel/comet assay applied to the
analysis of UV radiation-induced DNA damage in Rhodomonas sp. (Cryptophyta), Photochem. Photobiol. 74 (2001) 55–60.
[48] J.C. Ceballos, M.J. Bottino, H. Grossi Gallegos, Radiación solar en Argentina estimada por satélite: Algunas características espaciales y temporales, in: Divisão
de Satélites e Sistemas Ambientais – DSA, Centro de Previsão de Tempo e Estudos Climáticas – CPTEC, Instituto Nacional de Pesquisas Espaciais – INPE–Brasil,
División Física, Departamento de Ciencias Básicas, Universidad Nacional de
Luján, Argentina, 2005, p. 11.
[49] D. Schulte, R. Chabreck, Effects of nest and egg characteristics on size and early
development of American alligators, in: Proceedings 10th Working Meeting of
the Crocodile Specialist Group of the Species Survival Commission of IUCN –
The World Conservation Union, Gland, Switzerland, 1990, pp. 177–187.
[50] L.M. Verdade, Morphometric Analysis of the broad-snouted Caiman (Caiman
latirostris): an assessment of individual’s clutch, body size, sex, age, and area
of origin, Ph.D. Dissertation, University of Florida, Gainesville, FL, USA, 1997, p.
174.
[51] C.I. Piña, M. Simoncini, A. Larriera, Effects of two different incubation media on
hatching success, body mass, and length in Caiman latirostris, Aquaculture 246
(2005) 161–165.
[52] G.L Poletta, A. Larriera, E. Kleinsorge, M.D. Mudry, Genotoxicity of the herbicide formulation Roundup® (glyphosate) in broad-snouted caiman (Caiman
latirostris) evidenced by the Comet assay and the Micronucleus test, Mutat.
Res. 672 (2009) 95–102.
[53] M.A. Carballo, M.D. Mudry, Indicadores y Marcadores Biológicos, in: M.D.
Mudry, M.A. Carballo (Eds.), Genética Toxicológica, De los Cuatro Vientos,
Buenos Aires, Argentina, 2006, pp. 83–107.