Genotoxic activity of the commercial herbicide containing bifenox in bovine peripheral lymphocytes

Mutation Research 439 Ž1999. 129–135

Genotoxic activity of the commercial herbicide containing bifenox in bovine peripheral lymphocytes ˇ Katarına ´ Sivikova ´ ) , Jan ´ Dianovsky´ Department of Veterinary Genetics, UniÕersity of Veterinary Medicine, Komenskeho ´ 73, 041 81 Kosice, ˇ SloÕak Republic Received 21 July 1998; revised 8 December 1998; accepted 9 December 1998

Abstract The commercial herbicide with active element bifenox Žprincipal tradename Modown. was tested for the evaluation of genotoxicity in cultured cow peripheral lymphocytes in vitro. Several cytogenetic endpoints as chromosome aberrations ŽCA., sister chromatid exchanges ŽSCE., mitotic ŽMI. and proliferation ŽPI. indices were investigated in different sampling times. To detect possible metabolic modifications in herbicide genotoxicity, the cultures for SCE determination were also treated with S9 fraction. Cultures of lymphocytes were exposed to the herbicide at concentrations of 25, 50, 250, 500 and 1000 mgrml. A slight increase of CAs was found after exposure of this agent to doses ranging from 25 to 250 mgrml for 24 h. In the CA assay no statistical significance was seen. Both higher doses Ž500 and 1000 mgrml. caused a decrease of chromosome damage in comparison to the last active dose or control values correlated to induced cytotoxicity. Four concentrations Žall except the highest one. of the herbicide were applied into cultures in SCE assays both with and without metabolic activation. Significant elevations of SCE were observed after applications of herbicide tested at doses of 250 and 500 mgrml in each donor Ž P - 0.001 and P - 0.05, respectively. for 24 h. These concentrations also caused a statistically significant decrease in the MI and PI. Treatment for 48 h provided inadequate evidence for the genotoxic activity of the herbicide. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Commercial herbicide bifenox; Bovine peripheral lymphocyte; Chromosome aberration; Sister chromatid exchange; Cytotoxicity; Cell cycle delay

1. Introduction Herbicides represent a variable group of pesticides, extensively used at present in agriculture. Their wide spread, above all, the means of their application, directly into the soil, guarantee their

) Corresponding author. Fax: q421-95-63-23-666; E-mail: [email protected]

presence in the environment and thus they represent a potential risk to human genetic material. Bifenox is a selective herbicide used in control of annual broad-leaved weeds and some grasses in numerous crops, e.g., in cereals, maize, soya beans, rice and other crops. It acts by cellular membrane disruption and by inhibition of photosynthesis. It is often used in combination with other herbicides to extend the spectrum of activity. In this work, cultured bovine peripheral lymphocytes were used for the evaluation of genotoxic

1383-5718r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 1 8 Ž 9 8 . 0 0 1 8 4 - 3

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ˇ K. SiÕikoÕa, ´ J. DianoÕskyr ´ Mutation Research 439 (1999) 129–135

activity of commercial herbicide—with the active component of bifenox Žprincipal tradename Modown.. Genotoxic effects are considered among the most serious of the possible potential side effects of agricultural agents. Interactions of the chemical compounds with nuclear DNA are usually mutagenic or carcinogenic to the exposed organism w1,2x. Clastogenic or genotoxic effects of some groups of herbicides were demonstrated in human peripheral lymphocytes in vitro w3,4x. There are no data regarding the mutagenic or genotoxic potential of bifenox. Only short reports deal with its mammalian toxicity. Cattle are known as very sensitive animal species to the exposure to various environmental pollutants. There is evidence that they can accumulate xenobiotics in the body w5x. As cattle represent an important component of the human food chain, our results from cytogenetic studies using cytogenetic endpoints as chromosome aberrations ŽCA., sister chromatid exchanges ŽSCE., the reduction of mitotic ŽMI. and proliferation ŽPI. indices could be valuable information about the possibility of the genetic risk increase for humans.

2. Material and methods The commercial herbicide Modown Žconsists of bifenox, C 14 H 9 Cl 2 NO5 , 42%, and 58% inert components—composition not specified, Rhone-Poulenc, ˆ Toulouse, France. was dissolved into dimethyl sulfoxide ŽDMSO, Sigma, St. Louis, MO, USA. and applied into culture flasks. Concentrations of 25, 50, 250, 500 and 1000 mgrml Žexcept the highest dose for SCE assays. were used for treatments. The herbicide dose levels were chosen taking into account the highest doses for testing Ž500 and 1000 mgrml, respectively. causing a reduction in mitotic index ŽMI. ) 50%. The final DMSO concentration in the treated and control cultures was 0.1%. Ethylmethanesulfonate Ž EMS, Sigma; 250 mgrml., mitomycin C ŽMMC, Sigma; 0.4 mM. and cyclophosphamide ŽCPA, Jenapharm, Ankerwerk, Rudolstadt, Germany; 4 mgrml. were used as positive control agents in assays with absence and presence of the metabolic activation ŽS9 mix., respectively.

Experiments were carried out with two healthy cow donors ŽSlovak spotted cattle, 2 and 3 years old.. For SCE assays, experiments were made both in the presence and in the absence of the S9 mix. A freshly prepared S9 fraction Ž10% of the culture volume. from Aroclor 1254 ŽSupelco, Bellefonte, PA, USA. induced male mice was prepared according to the method of Maron and Ames w6x and applied to the control and experimental cultures. Glucoso-6-phosphate ŽAldrich, Milwaukee, WI, USA. and NADP ŽSigma. as cofactors were used. 2.1. Cell cultiÕation Whole blood cultures Ž0.5 ml. were cultivated for 72 h at 388C in 5 ml of RPMI 1640 medium supplemented with L-glutamine, 15 mMrl HEPES ŽSigma., 15% foetal calf serum, antibiotics Žpenicillin 250 Urml and streptomycin 250 mgrml., and phytohaemagglutinin ŽPHA, 180 mgrml, Wellcome, Darford, England.. Chromosome preparations for CA and SCE assays were obtained by the standard cytogenetic method; 2 h before harvest, colchicine ŽMerck, Darmstadt, Germany. was added at a concentration of 5 mgrml. Lymphocyte cultures without S9 fraction were exposed to the herbicide Modown for the last 24 and 48 h of cultivation, respectively, while cultures with S9 were treated with the herbicide for 2 h, then the cells were removed two times with medium and replaced in the complete fresh culture medium. For the SCE assay and the cell cycle kinetics bromodeoxyuridine Ž8 mgrml, BrdUrd, Sigma. was added to all cultures 24 h after initiation of division. Slides were stained with Giemsa for CAs or FPG technique to differentiate sister chromatids and cell cycles w7x. A total of 100 well-spread metaphases were analyzed for CAs; chromatid, isochromatid breaks and also gaps; chromatid and isochromatid exchanges ŽCB, CE, IB and IE., were also determined. A total of 50 differentially stained metaphases per donor and concentrations were examined for SCE, and 100 metaphases were analyzed for determination of M 1 , M 2 and M 3q mitotic divisions. Statistical analysis of results was performed using simple analysis of variance ŽANOVA., and then the

ˇ K. SiÕikoÕa, ´ J. DianoÕskyr ´ Mutation Research 439 (1999) 129–135

131

25 to 250 mgrml in each donor. The maximum frequency of breaks Žfour- or five-fold greater than corresponding controls. was found after exposure to the later dose. With respect to this dose, both highest tested concentrations Ž500 and 1000 mgrml, respectively. showed a decrease in the frequency of chromosome damage. Also, a significant inhibition in mitotic activity Ž P - 0.05, or P - 0.001, x 2-test. as well as a decrease in total number of cells in comparison to the controls were seen. In donor 2 a weak inhibition of MI Ž P - 0.05. was obtained after herbicide exposure to 50 mgrml. In the assay at 48 h, positive clastogenic effect was observed after treatment with the herbicide at a dose of 50 mgrml in donor 2, but an insufficient number of cells Ž67 metaphases. could be analyzed, the only common types of aberrations detected were chromatid breaks. At herbicide exposure to 250 and

Student’s t-test was applied to evaluate SCE occurrence between treated and untreated groups, and also for estimation of the cell cycle delay. A x 2-test was appropriated to estimate the induction of CAs and reduction of MI. Gaps were excluded from the total number of CAs and considered separately.

3. Results The frequency of chromosomal aberrations induced by the herbicide in bovine peripheral lymphocyte in vitro is shown in Table 1. No positive clastogenic effect was observed in the experiment with herbicide ŽModown. treatment at 24 h. A small elevation of induced chromosome damage was obtained when exposure to concentrations ranged from

Table 1 Induction of CAs in cow peripheral lymphocytes exposed to the commercial herbicide containing bifenox ŽModown. for 24 and 48 h Dose

Treatment for 24 h

Treatment for 48 h

Types of chromosomal aberrations G Donor 1 Control ŽDMSO. Herbicide Žmgrml. 25 50 250 500 1000 EMS Ž250 mgrml. Donor 2 Control ŽDMSO. Herbicide Žmgrml. 25 50 250 500 1000 EMS Ž250 mgrml.

CB

IB

CE

IE

2

2

–

–

–

4 8 8 9 5 11

1 5 6 3 0 4

1 – 2 2 – 3

– – – – – 2

– – – – – 3

6

3

1

–

–

4 7 9 8 4 10

3 4 10 4 2 9

1 3 1 – 1 1

– – – – – 1

– – – – – 4

Percent Ž%. breaks Ž"SD. 2.0 " 0.14 2.0 " 0.14 a 5.0 " 0.218 a 8.0 " 0.271a 5.0 " 0.218 a 0.0 " 0.0 ab 17.0 " 0.49)))

4.0 " 0.196 4.0 " 0.196 a 7.0 " 0.292 a 11.0 " 0.343 a 4.0 " 0.038 a 3.2 " 0.196 ac 20.0 " 0.678)))

Percent MI Ž%.

Types of chromosomal aberrations G

CB

IB

CE

IE

Percent Ž%. breaks Ž"SD.

Percent MI Ž%.

4.6

12

2

–

–

–

2.0 " 0.14

2.6

5.7 a 4.4 a 3.8 a 2.9) 1.3))) 2.3))

6 7 – – – 20

4 7 1 – – 10

1 – – – – 4

– – – – – –

– – – – – –

5.0 " 0.21a 8.5 " 0.28 ad ND ND ND 14.0 " 0.35))

2.3 a 1.5a 0.01))) 0 0 1.8 a

4.8

10

3

–

–

–

3.0 " 0.17

2.3

4.2 a 4.3 a 2.9) 1.6))) 0.8))) 2.9)

10 8 2 2 – 13

8 7 1 2 – 13

– – 1 – – –

– – – – – 1

– – – – – –

8.0 " 0.27 a 10.0 " 0.30)),e ND ND ND 15.0 " 0.36))

2.0 a 1.3 a 0.2))) 0.8)) 0.5))) 2.0 a

A total of 100 well-spread metaphases of each concentration was determined, if it was possible. )))Statistical significance Ž P - 0.001. — x 2 -test. a No statistically significant data. b 75, c 90, d 82, e 67 analyzed metaphases. ŽInsufficient number of cells.. ND—not done, CB, IB—chromatid, isochromatid breaks, CE, IE—chromatid, isochromatid exchange, G—gaps, not included into statistical data.

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ˇ K. SiÕikoÕa, ´ J. DianoÕskyr ´ Mutation Research 439 (1999) 129–135

500 mgrml, respectively, an apparent or total inhibition in mitotic activity was shown, caused by cellular membrane disruption and leakage of cells. The results obtained from the SCE assays and the lymphocyte proliferation kinetics are summarised in Tables 2 and 3. In Tables 2 and 3 the data obtained from all experimental conditions are shown: treatments for both 24 and 48 h without S9, and, respectively, for 2 h with S9, are included. A 24-h exposure to the herbicide Modown in the absence of S9 showed statistically significant elevations in the mean of SCEs in each donor at the highest concentrations tested Ž250 and 500 mgrml, P - 0.001 or P - 0.05, respectively, by ANOVA and Student’s t-test.. In conformity to results of the CA assay, the maximum frequency of exchange was observed at a dose of 250 mgrml. Both highest concentrations are reflected in the reduction of the PI ŽStudent’s t-test, P - 0.001 and P - 0.01, respectively.. A dose of 50 mgrml caused either a statisti-

cally significant increase in SCE frequencies or the reduction of PI in donors Ž P - 0.05.. Inadequate results for confirmation of genotoxicity of the herbicide were obtained in the prolonged sampling time. Treatment with the herbicide for 48 h caused a significant increase in SCE frequencies at a dose of 50 mgrml Ž P - 0.01. in both donors, but an insufficient number of second metaphases was analyzed Ž25 or 20, respectively.. All higher concentrations caused lethality of cells. A similar dose-dependence was found, but with lower frequencies of SCE in the treatment for 2 h with S9 microsomal fraction than in those seen for 24 h. Also, the largest increase in exchange frequencies was obtained after treatment with a dose of 250 mgrml Ž P - 0.01 or P - 0.001.. This dose also had a weak positive effect in reduction of cell cycle kinetics in donor 2. A weak positive effect on the induction of SCEs was observed after exposure to the herbicide at the dose of 500 or 50 mgrml

Table 2 Frequency of SCEs and PIs in cultured peripheral lymphocytes exposed to the commercial herbicide containing bifenox ŽModown. for 24 and 48 h without S9 Dose

Treatment for 24 h SCErcell

Donor 1 Control ŽDMSO. Herbicide Žmgrml. 24 h ŽyS9. 25 50 250 500 Positive control Ž0.4 mM MMC. Donor 2 Control ŽDMSO. Herbicide Žmgrml. 24 h ŽyS9. 25 50 250 500 Positive control Ž0.4 mM MMC.

Treatment for 48 h PI

SCErcell

PI

1.8

7.22 " 1.60

1.72

1.78 a 1.72 a 1.45))) 1.52)) 1.85a

7.56 " 2.63 a 8.92 " 1.82)),b ND ND 24.18 " 6.84)))

1.57 a 1.57 a

7.62 " 1.84

1.91

7.38 " 1.96

1.76

7.86 " 2.55a 8.02 " 2.40 a 9.95 " 3.23))) 8.78 " 1.87) 9.74 " 2.76)))

1.88 a 1.66) 1.51))) 1.53))) 1.75a

8.28 " 2.65a 9.25 " 2.40)),c 8.23 " 2.72 a,d ND 27.08 " 7.72)))

1.76 a 1.56 a 1.58 a

7.30 " 1.91

7.84 " 2.39 a 8.06 " 2.94) 10.46 " 3.08))) 8.38 " 2.28) 9.86 " 2.30)))

A total of 50 second-division metaphases of each group were analyzed for SCE, if it was possible. ), )), )))Statistically significant data Ž P - 0.05, P - 0.01, P - 0.001, ANOVA, Student’s t-test. a No statistical significance. b,c,d Insufficient number of second metaphases Ž25, 20, 17, respectively.. ND—not done.

1.57 a

1.55)

ˇ K. SiÕikoÕa, ´ J. DianoÕskyr ´ Mutation Research 439 (1999) 129–135 Table 3 Frequency of SCEs and PIs in cultured peripheral lymphocytes exposed to the commercial herbicide containing bifenox ŽModown. with S9 for 2 h Treatment for 2 h Donor 1 Control negative Control ŽDMSO. Herbicide Žmgrml. 2 h ŽqS9. 25 50 250 500 Positive control Ž4 mgrml cyclophosphamide. Donor 2 Control negative Control ŽDMSO. Herbicide Žmgrml. 2 h ŽqS9. 25 50 250 500 Positive control Ž4 mgrml cyclophosphamide.

SCErcell

PI

7.60"1.51 7.74"2.15

1.81 1.82

8.00"2.99 a 8.72"2.08) 9.20"2.25)) 8.76"2.41) 19.53"3.65)))

1.78 a 1.75a 1.64 a 1.76 a 1.82 a

7.50"1.86 7.56"2.16

1.74 1.82

7.22"1.12 a 8.18"2.10 a 8.96"1.77))) 8.64"2.52) 21.84"3.82 )))

1.72 a 1.76 a 1.58) 1.75a 1.83 a

A total of 50 second-division metaphases of each group were analyzed for SCE. ), )), )))Statistically significant data Ž P - 0.05, P - 0.01, P - 0.001, ANOVA, Student’s t-test.. a No statistical significance.

Ž P - 0.05., while a later dose was positive only in cultures of donor 1.

4. Discussion The herbicide Modown tested in bovine peripheral lymphocytes was shown to induce different spectra of cellular lesions, which led partly to SCE formation and partly to cell cycle delay and to the death of cells. From our results with the CA assay for 24 h, when gaps were excluded from statistical data, no clastogenic activity of the herbicide tested was found. A slight elevation of induced CA was seen after treatment with herbicide concentrations ranging from

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25 to 250 mgrml. Both doses of 500 and 1000 mgrml of the tested herbicide in our experiments induced a decrease in the frequency of chromosome alterations that was related to the inhibition of MI. The later dose caused a reduction of total cell number. CA are generally considered as biological endpoints to determine the level of genetic damage w8x. Statistically significant induced CA were described in human peripheral lymphocytes after treatment with herbicides of alachlor and maleic hydrazid w3x. No clastogenic effect was obtained with the herbicide trifluralin w4x. These negative observations were also correlated with induced cytotoxicity. Highly positive dose-related response in induced chromosome damage was seen in the SCE assays. A significant effect of the herbicide on SCE increase and on cell cycle kinetics was found in the treatment for 24 h without S9 at a dose of 250 mgrml Ž P - 0.001, Student’s t-test.. When compared to this dose, the highest concentration Ž500 mgrml. caused a decrease in the mean of SCEs reached at a lower significant value than those reached with the previous dose. This concentration also revealed a delay of cell division that was not much different than that seen with the previous dose Ž P - 0.001 or 0.01.. The treatments with the herbicide for 2 h with S9 had no positive effect on the cell cycle delay Žwith the exception of a dose of 250 mgrml in donor 2.. The frequencies of induced exchange were somewhat lower than those seen for 24 h Žreached with the same or minor statistical significance.. The lower activity of the herbicide tested seems to be caused by the reduction of time-exposure. Our results from the SCE assay correspond with several other authors who have regarded SCEs as very sensitive bioindicators of genotoxic agents w8– 11x. The significant reduction of PI found at the highest concentration in our experiments for 24 h was probably caused by a decrease of more damaged cells w12x, and thus collections of less damage cells could be observed. The downward-bending phenomenon of the dose–response curve after treatments with increasing concentrations of alkylating mutagens in human peripheral lymphocytes was described by Morimoto et al. w10x. Also Puig et al. w13x explained for example a small increase in CA frequencies after exposure to the pyrethroid insecticide

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ˇ K. SiÕikoÕa, ´ J. DianoÕskyr ´ Mutation Research 439 (1999) 129–135

cypermethrin through the induction of statistically significant cell division delay. Similarly, Scarpato et al. w14x reported that highly damaged cells, which enter into mitoses, were delayed or failed to occur; this might mask the expression of genotoxic effects. The reduction of MI and PI represent an additional check on the chemical agent potency that can be interpreted as cell cycle delays andror in terms of induced cytotoxicity w15–17x. A more expressive cytotoxic effect was found with the prolonged time sampling of herbicide incubation. With respect to found cell proliferation delay, treatment for 48-h incubation was provided to confirm evidence of herbicide genotoxicity. All higher concentrations tested Ž250, 500 and 1000 mgrml. both in CA and SCE assays for 48 h appeared through cellular membrane disruption and caused lethality of cells. Bifenox is a member of a group of nitrophenylether herbicides known to act by producing reactive radical species in plants that initiate destructive reactions in chloroplast membrane lipids leading to death of cells w18x. As far as we aware there is no information of cytogenetic endpoints presented here regarding human or animal cells exposed to bifenox. Either a direct mutagenic effect on the DNA or an alternatively indirect mutagenic effect via enzymatic modification for chemical agents should be considered w19,20x. SCEs are efficiently induced by those substances that form covalent adducts to the DNA or otherwise interfere with DNA metabolism or repair. On the other hand, substances that directly break the backbone of DNA w21x efficiently induce chromosome breaks. When extranuclear lesions can be included into the induction of SCEs it just could have been an explanation for greater sensitivity of this assay in comparison with the CA induction. In conclusion, our results indicate that the herbicide tested is able to exert clear cytotoxic and genotoxic effects in bovine peripheral lymphocytes.

Acknowledgements This work was supported by the Ministry of Education and Science of the Slovak Republic ŽGrant No. 1r5150r98 and Grant No. 4342r1997..

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