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Mutation Research 629 (2007) 81–88

Assessment of the genotoxicity in toad Bufo raddei exposed topetrochemical contaminants in Lanzhou Region, China

Dejun Huang a, Yingmei Zhang a,∗, Yejing Wang a, Zhuoyi Xie a, Weihong Ji b

a School of Life Sciences, Lanzhou University, Lanzhou 730000, PR Chinab Institute of Natural Resources, Massey University, Private Bag 102 904, North Shore Mail Centre, Auckland, New Zealand

Received 24 August 2006; received in revised form 20 December 2006; accepted 16 January 2007Available online 2 February 2007

bstract

Single cell gel electrophoresis or comet assay, micronucleus (MN) test and global DNA methylation detection were used to assesshe genotoxicity in toad Bufo raddei exposed to the petrochemical (mainly oil and phenol) polluted area in Lanzhou Region (LZR)omparing with a relatively unpolluted area in Liujiaxia Region (LJXR). The results from the present study indicated that DNAamage and MN frequency in toad from LZR were significantly higher than those from LJXR at the same sampling month, whereashe degree of global DNA methylation was lower, which implies that the petrochemical contaminants at environmental level in LZRere genotoxic to B. raddei. The degree of genotoxic damage was obviously related with the extent of pollution among the three

ampling months in LZR. The significantly positive correlations between DNA damage and concentrations of oil and/or phenol

xisted in liver cells but erythrocytes, implying that liver is more suitable as a sentinel tissue for the assessment of genotoxic impactf low-level contamination. The results from both comet assay and global DNA methylation detection on liver cells showed that theenotoxicity varied significantly with oil and/or phenol concentrations, suggesting that these two methods are relatively sensitivend suitable for monitoring the genotoxicity of petrochemical pollutants on amphibians.

2007 Published by Elsevier B.V.

toring;

eywords: Petroleum contamination; Amphibian; Genotoxicity moni

. Introduction

Single cell gel electrophoresis or comet assay isainly used to detect DNA strand breaks and alkali labile

ites. It is a very sensitive and fast method for measuringNA damage [1–4]. Micronucleus (MN) assay allows

he detection of both chromosome fragments as well as

hole chromosomes which are left behind when chro-osomes emigrate to the poles in anaphase, and in recent

ears it has been also used to monitor the genotoxicity

∗ Corresponding author. Tel.: +86 13919123067;ax: +86 931 8913631.

E-mail address: ymzhang@lzu.edu.cn (Y. Zhang).

383-5718/$ – see front matter © 2007 Published by Elsevier B.V.doi:10.1016/j.mrgentox.2007.01.007

DNA damage

of many chemicals [5–8]. The degree of global DNAmethylation may be also a good biomarker for genotox-icity assessment because global DNA hypomethylationhas been proposed to be involved in chemical carcino-genesis [9–11].

With the development of industry, petroleum hasbeen recognized as an important part of environmen-tal contaminants since shortly after the beginning of thetwentieth century [12]. In areas of petroleum industry,crude oils (mainly polycyclic aromatic hydrocarbons,PAHs) and phenols are the major fractions. Previous

studies [3,4,13–16] have shown that both PAHs and phe-nols could induce the genotoxicity effects in animalsand human beings, even at environmental level of lowconcentrations [17–20].

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82 D. Huang et al. / Mutati

This research has been conducted in Lanzhou Region(LZR, E103◦40′, N35◦54′) and Liujiaxia Region (LJXR,E103◦27′, N35◦47′), northwest of China. The YellowRiver flows through LJXR and LZR from west to eastand is the most important source of water supply inthese two regions. Oil refinery and chemical mills are theprimary industries in LZR. LJXR is a relatively unpol-luted upstream area, only 75 km southwest of LZR withsimilar organism components and other natural charac-teristics to LZR. The industrial wastes (mainly oil andphenol) from the refineries and factories in LZR haveheavily polluted the aquatic environment of the YellowRiver and may have inevitably affected the organismsliving in it. However few reports can be found regardingthe effects of the petrochemical pollutants on the nativespecies in these regions.

Amphibians are more susceptible to environmentalcontaminants compared with birds and mammals fortheir permeable skin which can more easily absorb sur-rounding moisture and substances dissolved in water.This study focuses on Bufo raddei, the most commontoad species in LZR and LJXR. The aim of the study isto investigate:

(1) Which one among comet assay, micronucleus testand global DNA methylation assay is the most sensi-tive method for assessing the potential genotoxicityof the petrochemical pollutants.

(2) Which one between blood and liver is a better mate-rial for detecting genotoxicity.

2. Materials and methods

2.1. Chemicals

Cytosine (C), 5-methylcytosine (5-MeC), DNAase-freeRNAase, proteinase K and SDS, low and normal melting point

Fig. 1. Map showing the location of Liujiaxia Re

arch 629 (2007) 81–88

agaroses were obtained from Sigma (USA). Sodium lauroylsarcosine, Tirton X-100 and dimethyl sulfoxide (DMSO) wereproduced by Amresco. All other chemicals and reagents wereof analytical grade and obtained from local chemical compa-nies.

2.2. Sample collection

Specimens of toad B. raddei were collected from lentichabitats (ponds and wetlands) along the Yellow River in LJXRand LZR (Fig. 1) during April, June and August in the year of2004 and 2005. During each sampling month, 18 adult toads(10 males and 8 females) with similar body size (body weight:13.15–14.8 g, body length: 48.5–54.5 cm) were captured fromeach sampling area. The water in the ponds and wetlands wasalso sampled to determine the concentrations of the major pol-lutants (oil and phenol) using the national standard method ofwater analysis (GB/T 16488-1996, GB 7490-87). The capturedtoads were euthanized by pithing [21] in laboratory. Once thetoad was confirmed dead, blood was immediately collected forcomet assay and MN test, and liver for comet assay and globalDNA methylation detection.

2.3. Comet assay

2.3.1. Preparation of single cell suspension andmicroscope slide

The erythrocytes in blood were collected by centrifugation,directly diluted in PBS buffer. The liver was isolated, rinsedin Hank’s balanced salt solution, and then minced into finepieces to obtain a cell suspension. The erythrocytes and livercells were diluted so that three or four cells would be seen in asingle field at 400× magnification. The resulting cell suspen-sion (cell viability was found to be over 90% by the Trypan

Blue exclusion analysis) was mixed with 0.6% low meltingpoint agarose (kept in a 37 ◦C water bath). This mixture wasthen placed onto a thin layer of agarose (with normal melt-ing point) supported by a frosted microscope slide and thencovered with a 24 mm × 24 mm coverslip.

gion (LJXR) and Lanzhou Region (LZR).

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.3.2. Electrophoresis and analysisThe procedure used was originally described by Singh et

l. [22] and modified by Hartmann et al. [23]. After gentlyemoving the coverslip, the slide was submersed in lysing solu-ion (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, pH 10, 1%odium lauroyl sarcosine, 10% dimethl sulfoxid and 1% Tri-on X-100 added just before use, 4 ◦C) for 2 h and then werelaced in the electrophoresis solution (300 mM NaOH, 1 mMDTA, pH > 13, 4 ◦C) for 20 min to allow DNA unwinding.lectrophoresis was conducted for 20 min at 25 V and 300 mA.fter electrophoresis, the slides were drained and neutralized

or 3 × 5 min with neutralization buffer (0.4 M Tris–HCl, pH.5) and stained with ethidium bromide (5 �g/ml).

The nucleoids were scored visually using a fluorescenceicroscope (Nikon) with an excitation filter of 510–560 nm andbarrier filter of 590 nm at 400× magnification. The degree ofNA damage was analyzed using the method by Andrighetti-rohner et al. [24] with some modifications. Fifty comet cellsrom each coded slide (two slides per sample) scored by each ofhe three authors at random (in the central area of each codedlide) were classified into five classes based on the shape ofhe comets: type 1, comets with no tail; type 2, with small tailtail length is less than a quarter of the head diameter); type 3,ith medium tail (tail length is in between a quarter and a headiameter); type 4, with long tail (tail length is greater thanead diameter); type 5, comets with poorly defined or smallead. Numerical values of 0–4 were assigned to the cometypes 1–5, respectively. The product of the numerical valuesas calculated for each sample and used for the analysis. The

otal score for 50 comet cells could range, therefore, from 0all undamaged) to 200 (all maximally damaged). The degreef DNA damage was presented as the percentage of DNA inail.

.4. Micronucleus test

The erythrocytes cells were fixed with methanol/acetic acid3:1) plus 0.75% of formaldehyde for 10 min and dropped onlass slides (two slides per toad, 18 toads per region per sam-ling time). Then the coded slides were dyed with Giemsa4%, pH 6.98) for 15 min, washed several times with distilledater, imaged with a Nikon microscope under oil immersion

ens (1000× magnification) and micronucleus frequency (notncluding the nucleus whose chromosomes are in anaphase)as recorded as permillage. Five thousand erythrocytes per

lide and two slides per animal were scored separately by threeuthors, and the mean values were used in analysis.

.5. Global DNA methylation assay

The isolation of DNA in liver tissue and global DNA methy-

ation assay were conducted as previously described [9]. A.2 g liver tissue was homogenized in 0.5 ml TE buffer (10 mMris–HCl, pH 8.0, 0.1 M EDTA) containing 0.5% SDS, and

hen the homogenate was treated with DNase-free RNase200 �g/ml) and proteinase K (200 �g/ml) and then extracted

arch 629 (2007) 81–88 83

three times with phenol, once with phenol:chloroform (1:1),and finally with chloroform. The DNA was precipitated withcold ethanol containing 10 M ammonium acetate (10% vol.),washed twice with 70% ethanol, and hydrolyzed in 100 �l of12 M perchloric acid at 100 ◦C for 1 h. After the addition of230 �l of 6 M KOH, the precipitate was removed by centrifuga-tion. The supernatant filtered through a 0.2 �m polypropylenesyringe filter (Whatman Inc., Clifton, NJ) and analyzed usinga HPLC system equipped with a Whatman Partisphere C18column (4.6 mm × 250 mm, 5 �m particle). The column waseluted for 15 min with an isocratic mobile phase of 100 mMammonium acetate (pH 4.25) containing 0.5% acetonitrile.The flow rate was 2 ml/min and the detection wavelengthwas 280 nm. The retention time for cytosine and 5-MeC were5.0 and 6.5 min, respectively. The percentage of global DNAmethylation presented as 5-MeC was calculated from the peakareas using the following formula:

5-MeC

(5 − MeC + cytosine)× 100.

2.6. Statistical analysis

The results were expressed as means ± standard errors(S.E.s). The analysis about the concentrations of oil and phe-nol in LZR and LJXR compared with those of environmentalquality standard III for surface water in China was performedwith a one-sample t-test, and the comparison between the twosampling areas was performed with a one-tailed Student’s t-test. Pearson correlation test was used to detect the correlationbetween genotoxicity and pollutant concentrations (p < 0.05is accepted as statistically significant, and p < 0.01 as greatlysignificant).

3. Results

Concentrations of the major pollutants (oil and phe-nol) in LJXR and LZR were detected and summarizedin Table 1. And their concentrations in LZR were higherthan those of environmental quality standard III for sur-face water in China (GB 3838-2002) during the threesampling months, and the differences were significant inApril (p < 0.01) and June (p < 0.05). However, the con-centrations in LJXR were lower than the threshold of thequality standard III (Oil: 0.03 mg/l; Phenol: 0.005 mg/l).

We have observed five classes of comets from thecomet assay (Fig. 2). DNA damage in erythrocytes andliver cells, and MN frequency in erythrocytes fromLZR were significantly higher than those from LJXR(p < 0.05–0.01). The degree of global DNA methylation

in the toad liver from LZR was significantly lower thanthat from LJXR (p < 0.05). Meanwhile, enhanced DNAdamage and MN frequency, and reduced global DNAmethylation appeared in the highest polluted month,

84 D. Huang et al. / Mutation Research 629 (2007) 81–88

Table 1The concentrations (Mean ± S.E.) of the major pollutants in LJXR and LZR

April (n = 12)a June (n = 12)a August (n = 12)a

LJXRb LZRb LJXRb LZRb LJXRb LZRb

Oil concentration (mg/l) 0.03c 0.02 ± 0.00 0.06 ± 0.01* 0.02 ± 0.00 0.05 ± 0.01d,e 0.02 ± 0.00 0.04 ± 0.01**,f

Phenol concentration (mg/l) 0.005c ND 0.008 ± 0.001* ND 0.006 ± 0.001d,** ND 0.005 ± 0.001**,f

ND: Not detectable.a Sampling period.b Sampling area.c The threshold values of environmental quality standard III for surface water in China.d p < 0.05.e p < 0.05.

d III for

f p < 0.05 compared with the value in June.* p < 0.01compared with the value of environmental quality standar

** p < 0.01compared with the values in April.

April. The genotoxicity was relatively light in Augustwhen the degree of pollution is lowest (Table 2).

DNA damage, MN frequency and the degree of globalDNA methylation in toad from LZR were significantlycorrelated to the concentrations of oil and/or phenol(Figs. 3 and 4). DNA damage in liver cells was signifi-cantly positive-correlated with the concentrations of oiland/or phenol (p < 0.01), while the degree of global DNAmethylation was negative-correlated (p < 0.01). How-

ever, DNA damage in erythrocytes was not significantlycorrelated with the concentrations of oil and/or phe-nol (p > 0.05). MN frequency of erythrocytes showeda significant negative-correlation with oil concentration

Fig. 2. The five classes of the comets: a, no tail; b, comet with small tail (taitail (tail length between a quarter and a full of head diameter); d, comet with ldefined or small head (magnification: 200×).

surface water in China.

(p < 0.05). Although it showed the same trend, the cor-relation between MN frequency of erythrocytes andphenol concentrations was not statistically significant(p > 0.05).

4. Discussion

The use of biomarkers in genotoxicology to eval-uate the environmental quality has become a major

tool in environmental risk assessment. Studies havebeen conducted on genotoxicity of inorganic [25],organic [3,26,27] and mixed [7,28] pollutants on aquaticorganisms such as mussel [3], shrimp [29] and fish

l length less than a quarter of head diameter); c, comet with mediumong tail (tail length greater than head diameter); e, comet with poorly

D. Huang et al. / Mutation Research 629 (2007) 81–88 85

Table 2DNA damage, micronucleus frequency and the degree of global DNA methylation (Mean ± S.E.) in B. raddei from LJXR and LZR during differentsampling periods

April (n = 18)a June (n = 18)a August (n = 18)a

LJXRb LZRb LJXRb LZRb LJXRb LZRb

DNA damage inerythrocytes (%)c

4.74 ± 0.46 9.13 ± 0.72* 4.46 ± 0.48 9.37 ± 0.65*,e 5.53 ± 0.41**,*** 8.65 ± 0.63d,**,***

DNA damage in liver(%)c

6.38 ± 0.48 18.21 + 1.05* 6.47 ± 0.66 6.87 ± 1.33*,** 5.98 ± 0.54 12.20 ± 0.87d,**,***

MN frequency inerythrocytes (‰)

3.06 ± 1.10 8.16 ± 2.04* 3.44 ± 1.02e 8.73 ± 2.41*,** 2.18 ± 1.10**,*** 8.29 ± 2.56*,e,***

The degree of globalDNA methyllation inliver

77.01 ± 2.76 65.71 ± 3.18d 66.62 ± 2.63** 61.64 ± 2.94d,e 94.52 ± 4.26**,*** 89.31 ± 3.74d,**,***

a Sampling period.b Sampling area.c DNA damage was presented as the percentage of DNA in tail.d p < 0.05.e p < 0.05.* p < 0.01 when the values in LZR compared with those in LJXR in the same sampling periods.

** p < 0.01 compared with the values in April.*** p < 0.01 compared with the values in June.

Fig. 3. Correlation between oil concentration (0.04 mg/l in August,0.05 mg/l in June and 0.06 mg/l in April in LZR) and DNA damage (a)in erythrocytes and liver cells, MN frequency in erythrocytes (b), thedegree of global DNA methylation in liver (c) of toad in LZR (n = 18).

Fig. 4. Correlation between phenol concentration (0.005mg/l inAugust, 0.006 mg/l in June and 0.008 mg/l in April in LZR) and DNAdamage (a) in erythrocytes and liver cells, MN frequency in erythro-cytes (b), the degree of global DNA methylation in liver (c) of toad inLZR (n = 18).

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[7,28,30,31]. As for amphibians, the genotoxicity ofherbicide [26], pesticide [27,32], polluted river water[33–35], aqueous extracts of soils, bottom ash and sedi-ments [36,37] have been also studied by using MN andcomet assay. Petrochemicals such as oils, PAHs and phe-nols have been shown to have strong genotoxic potentialto oyster, mussel, fish and amphibian larvae [3,6,38–40].In this study, significant genotoxicity in adult B. raddeifrom LZR was observed through comet assay, MN testand global DNA methylation detection, which was inaccordance with the results of the previous studies men-tioned above. Our result suggests that the petrochemicalcontaminants were genotoxic to the toad B. raddei evenat concentrations far below the lethal level. However,genotoxicity of petrochemicals on B. raddei varied in thedifferent sampling periods, which suggests that the geno-toxic damage could be reduced by reducing the degreeof pollution.

Previous studies indicate that blood is a goodbiomarker for detecting genotoxicity and erythrocyteshave often been used to monitor DNA damage throughcomet assay [6,7,18]. In our study, comet assay onboth erythrocytes and liver cells showed a significantincreased DNA damage in toad from the polluted areaLZR. There is a significant positive-correlation betweenthe increased DNA damage in liver cells and the con-centrations of oil and/or phenol. However, the increasedDNA damage in erythrocytes showed no significant cor-relation with the concentrations of oil and/or phenol. Themicronucleus frequency in erythrocytes was not signifi-cantly correlated with phenol concentration. This resultsuggests that liver is a better biomarker than blood forassessing genotoxicity in B. raddei. We found significantnegative correlation between the frequency of MN andoil concentration. Similar results have been obtained inmany fish by Nepomuceno et al. [41], Ayllon and Garcia-Vazquez [42], and Rodriguez-Cea et al. [43]. This wasexplained by previous studies as results of differences incharacteristics and toxic effects of different chemicals, incombination of differences in the responses of differentfish species. To our knowledge, abnormal nuclear is theprecursors of MN. It occurs more often when combatingwith the toxicity [42,44,45]. With the decrease of MN intoad, it is likely to observe an increase in the frequencyof abnormal nuclear at low pollutant concentrations inthe environment.

Global DNA methylation detection showed thatthe decrease of global DNA methylation significantly

depended on oil and/or phenol concentrations, whichsuggested that both comet assay and global DNAmethylation detection were suitable for monitoring thegenotoxicity of pollutants on amphibians.

arch 629 (2007) 81–88

5. Conclusion

Our results from comet assay, MN test and globalDNA methylation detection indicate that the petrochem-ical pollutants were greatly genotoxic to the toad B.raddei. Significant positive correlations were detectedbetween DNA damage and oil and/or phenol concentra-tions in liver cells but not in erythrocytes. This suggeststhat liver, rather than blood, is more suitable as a sentineltissue for the assessment of genotoxic impact of low-level contamination. Our results also imply that cometassay and global DNA methylation detection on livercells are relatively sensitive and are suitable methodsfor monitoring the genotoxicity effects on amphibiansexposed to petrochemical pollutants.

Acknowledgements

This work was supported by National Natural Sci-ence Foundation of China (No. 30470320) and NaturalScience Foundation of Gansu Province, China (No.3ZS051-A25-084).

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