In Vitro Cytogenetic Results Supporting a DNA Nonreactive

May 23, 2008 - the eighties, the precise mechanism of action is still relatively undefined. At present, increasing evidence suggests that OTA does not...
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Chem. Res. Toxicol. 2008, 21, 1235–1243

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In Vitro Cytogenetic Results Supporting a DNA Nonreactive Mechanism for Ochratoxin A, Potentially Relevant for Its Carcinogenicity Pasquale Mosesso,*,† Serena Cinelli,‡ Joaquin Pin˜ero,§ Raffaela Bellacima,† and Gaetano Pepe† Dipartimento di Agrobiologia e Agrochimica, UniVersita` degli Studi della Tuscia, Via San Camillo de Lellis snc 01100 Viterbo, Italy, Research Toxicology Centre, Via Tito Speri 14, 00040 Pomezia (Roma), Italy, and Department of Cell Biology, UniVersity of SeVille, C/Reina Mercedes snc, 41012 SeVille, Spain ReceiVed January 18, 2008

Ochratoxin A (OTA) is a widespread mycotoxin of cereals and many agricultural products and causes high incidences of renal tumors in rodents. Although its carcinogenic properties have been known since the eighties, the precise mechanism of action is still relatively undefined. At present, increasing evidence suggests that OTA does not act with a direct genotoxic mechanism, opposed to other previous evidence where the formation of DNA adducts by 32P-postlabeling was observed. The genotoxic activity of OTA assessed in a variety of in vitro and in vivo studies was very low if genotoxic at all. In this study, we clearly show that OTA does not bear any clastogenic or aneugenic activity based on the absence of the induction of chromosome aberrations, sister chromatid exchanges, and micronuclei in human lymphocytes and V79 cells in vitro in both the absence and the presence of S9 metabolism. Alternatively, cytogenetic analyses evidenced significant increases in endoreduplicated cells and highly condensed metaphases with separated chromatids. This implies that OTA or its possible metabolites do not covalently bind DNA through the formation of adducts since structural chromosome aberrations are a very sensitive end points to detect chemical carcinogens with electrophilic substituents. Alternatively, induction of endoreduplication and chromatid separation provides strong evidence for a DNA nonreactive mechanism of OTA carcinogenicity involving the disruption of mitosis by interfering with key regulators of chromosome separation and progression of mitosis. This causes a temporary arrest of mitoses and premature exit from it (mitotic slippage) to generate endoreduplication and polyploidy accompanied by increased risk of aneuploidy and subsequent tumor formation. Introduction Ochratoxin A (OTA)1 is a mycotoxin produced by several species of fungi (Penicillium and Aspergillum species), and it occurs naturally in cereal and grain products. OTA is a nephrotoxic mycotoxin with strong carcinogenic properties to rodents (1, 2) and possesses teratogenic, immunotoxic (3–5), and possibly neurotoxic activities (6, 7). Furthermore, it has been considered potentially responsible for the development of nephropathies observed in different farm animals (8). In humans, it is supposed to be associated with Balkan endemic nephropathy (BEN) and is possibly involved in the origin of urinary tract tumors (9). On these bases, OTA has been classified as a possible human carcinogen by the International Agency for Research on Cancer (IARC) (10). Since then, the carcinogenic mechanism of OTA has still been relatively undefined, although at present increasing evidence suggests that OTA does not act with a direct genotoxic mechanism. This is supported by different studies with radiolabeled OTA unable to detect any * To whom correspondence should be addressed. Tel: +39 0761 357257. Fax: +39 0761 357257. E-mail: [email protected]. † Universita` degli Studi della Tuscia. ‡ Research Toxicology Centre. § University of Seville. 1 Abbreviations: OTA, ochratoxin A; SCEs, sister chromatid exchanges; CHO, Chinese hamster ovary; PHA, phytohemoagglutinin; BrdU, 5-bromo2′-deoxyuridine; MI, mitotic index; CBPI, cytokinesis-block proliferation index.

DNA binding of OTA (11–14) as opposed to previous studies using the 32P-postlabeling method in which DNA adducts were reported (15–17), although the authors did not provide structural information on the nature of the adduct. On the other hand, the genotoxic activity of OTA assessed in a variety of in vitro and in vivo studies was very low if genotoxic at all. OTA did not prove to be mutagenic in Salmonella typhimurium in both the absence and the presence of metabolic activation systems, when assays were conducted according to standard protocols (2, 18–22), although mutations after exposure to OTA were reported using nonvalidated modifications of the Ames test (23). OTA was also negative in a Salmonella microsome assay with a HepG2derived enzyme (S9 mix) with the TA98 and TA100 strains (24) and in the Escherichia coli SOS spot test at nontoxic concentrations (25–27). In mammalian cells, conflicting results were obtained regarding the potential of OTA to induce mutations. OTA was not mutagenic in thymidine kinase (TK() mouse lymphoma cells or in the hypoxanthine-guanine phosphoribosyltransferase (HPRT) test system (19, 22). In contrast, mutagenic effects of OTA were reported in NIH 3T3 mouse fibroblasts stably transfected with human cytochrome P450s (28), although these results are difficult to interpret since studies on OTA metabolism indicate that bioactivation does not play a role in OTA toxicity and carcinogenicity (11–13, 29, 30). In Chinese hamster ovary (CHO) cells, chromosomal aberrations were not seen after exposure to OTA, but only a small increase in the frequency of sister chromatid exchanges (SCEs) was noted

10.1021/tx800029f CCC: $40.75  2008 American Chemical Society Published on Web 05/23/2008

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(2). Unscheduled DNA repair synthesis was observed in several cell types in response to OTA treatment (31–33), and OTA induced DNA strand breaks independently of in vitro metabolic activation. However, some of these studies used extremely high concentrations of OTA, and the observed DNA damage may likely be a result of the high cytotoxicity (24, 28, 34–36). In in vivo studies, DNA strand breaks were observed in spleen, liver, and kidneys of mice treated with a single dose of OTA using the alkaline elution technique. More recently, DNA strand breaks, suggestive of oxidative DNA damage, were detected in rat kidneys in response to repeated administrations of OTA for 2 weeks using the Comet assay (37). In the present paper, to gather further information on a direct interaction of OTA or its metabolites with DNA, we generated a thorough in vitro cytogenetic profile in human lymphocytes and a V79 rodent cell line.

Materials and Methods Chemicals. OTA (CAS no. 303-47-9), 5-bromo-2′-deoxyuridine (BrdU), cytochalasin B, cyclophosphamide, and mitomycin-C were purchased from Sigma-Aldrich. All other chemicals and organic solvents were obtained from the same supplier at the highest purity available. OTA was prepared immediately before treatment in dimethylsulfoxide (DMSO) and added to the culture medium such that the final concentration of solvent did not exceed 1%. The positive control substances, cyclophosphamide and mitomycin-C, were dissolved in culture medium. Test Systems and Culture Conditions. Chinese hamster V79 cells were originally obtained from Dr. J. Williamson (British American Tobacco, United Kingdom). The karyotype, generation time, plating efficiency, and absence of mycoplasmal contamination were checked at regular intervals. Permanent stocks of the V79 cells were stored in liquid nitrogen, and subcultures were prepared from the frozen stocks for experimental use. Cultures of the cells were grown in Eagle’s minimal essential medium (EMEM) supplemented with 10% fetal calf serum, 2 mM L-glutamine, and antibiotics (1% w/v penicillin and 86 µM streptomycin). All incubations are at 37 °C in a 5% carbon dioxide atmosphere and 100% nominal humidity. Human lymphocytes were prepared using fresh venous blood drawn from a male healthy donor into heparinized vacutainers. Aliquots of whole blood (0.5 mL) were added to each 4.5 mL of RPMI-1640 supplemented with 20% heat-inactivated fetal calf serum (Gibco BRL), 2% w/v phytohemoagglutinin (PHA, Murex Diagnostics, Dartford, United Kingdom), 100 U/mL Hepes buffer, 2 mM L-glutamine, and antibiotics (1% w/v penicillin and 100 µM streptomycin). Cultures were incubated at 37 °C in the dark. Preparation of in Vitro Metabolic Activation System (S9). Rat liver and kidney S9 tissue homogenates were prepared from young male rats following mixed induction with betanaphtoflavone and phenobarbital. S9 tissue fractions were checked for protein content (Lowry method), aminopyrine demethylase activity, and finally for metabolizing capability in an Ames test with the indirect mutagens 2-aminoanthracene and benzo(a)pyrene using the S. typhimurium tester strain TA100 as displayed in Appendices 2a and 2b. The S9 mix was prepared with the following composition and was added to culture medium at 10% w/v final concentration: S9 fraction, 3.0 mL; NADP (0.1 M), 0.4 mL; G-6-P (0.1 M), 0.5 mL; KCl (0.33M), 1.0 mL; MgCl2 (0.2M), 0.4 mL; Hepes buffer (0.2M), 1.0 mL; and Hank’s saline, 3.7 mL, all of which equalled 10.0 mL.

Mosesso et al.

Chromosomal Aberration and SCEs Assays. Treatments with OTA were performed in both the absence and the presence of rat liver or kidney S9 metabolism in Chinese hamster V79 cells. Additional treatments in human lymphocytes from whole blood cultures (at 48 h from stimulation with PHA) in the absence and presence of rat liver S9 were also performed. The studies were designed to comply with the experimental methods indicated in the guidelines of: OECD Guidelines for the testing of chemicals no. 487 (draft, June 2004). Following dose-range finding experiments, the assays were performed using dose levels of 2476.4, 1149.0, 532.4, 247.6, 114.9, 53.2, and 24.8 µM evenly spaced at 1/5 log intervals for 3 h. The experiments also included solvent vehicle, untreated, and positive control treatments (mitomycin-C and cyclophosphamide in the absence and presence of S9 metabolism, respectively). At the end of treatment (3 h), cultures were washed twice with PBS solution and reincubated at 37 °C in fresh complete culture medium for further 18 h (approximately 1.5 cell cycle). Cultures set up for analysis of SCEs also received BrdU at 9.8 µM to differentiate sister chromatids. Colcemid at 0.27 µM was added during the last 3 h of culture to accumulate cells in the metaphase. Cytogenetic preparations from these cultures were obtained following standard procedures. The mitotic index (MI) was then scored for each dose level, and three dose levels for scoring of metaphases were selected for each of the different treatment series according to the reduction of MI observed. This procedure allowed the selection of cultures for scoring at the required level of toxicity and avoided problems resulting from the variability of cytotoxicity from experiment to experiment. The highest dose level selected for metaphase analysis should be a dose that produces a substantial reduction of MI as compared with the solvent controls. Ideally, the reduction should be approximately 50% and treatments reducing the MI below 20% should not be used. Three lower dose levels from the highest selected were also included in the analysis of chromosomal aberrations and SCEs. Scoring for chromosomal damage was undertaken blind with coded slides. A minimum of 100 metaphases per culture was scored for chromosomal aberrations and 50 second-division well-spread metaphases for SCEs. Chromosomal aberrations were classified as chromatid type gaps, chromatid type breaks, chromatid type exchanges, chromosome type gaps, chromosome type breaks, chromosome type exchanges, and isolocus events that include isochromatid and isolocus breaks when these cannot be distinguished, as described by ref 38. The experiment also included negative and positive controls. The negative control consisted of both untreated and solvent-treated cultures. The positive controls were cyclophosphamide and mitomycin-C in the presence and absence of S9 metabolism, respectively. For the chromosome aberration assay, the number of aberration-bearing cells (excluding gaps) was utilized for statistical analyses. To determine the statistical significance, Fisher’s exact test was used. A test substance was considered positive when statistically significant increases in aberration-bearing cells were observed at two consecutive dose levels or at the higher dose level and exceeded the historical control mean values. For the SCEs assay, a test substance producing neither a statistically significant dose-related increase in the mean number of SCEs per cell nor a statistically significant and reproducible positive response at any one of the test points employed evaluated by Student’s t test was considered negative. In Vitro Micronucleus Assay. Treatments with OTA were performed in both the absence and the presence of rat liver S9 metabolism in human lymphocytes from whole blood cultures.

DNA NonreactiVe Mechanism of Ochratoxin A

Following dose-range finding experiments, the assay was performed using dose levels of 247.6, 114.9, 53.2, 24.8, 11.5, 5.3, and 2.5 µM evenly spaced at 1/5 log intervals. The experiment included untreated, solvent, and positive control cultures. Positive control treatments included in the absence of S9 the clastogen mitomycin-C and the aneugen colchicine. In the presence of S9 metabolism, only the clastogen cyclophosphamide was used since no aneugen requiring metabolic activation was commonly recognized. Treatments were performed according to the experimental scheme displayed in Appendix 1. At the end of treatment time, the cell cultures were centrifuged and washed twice with PBS. Fresh medium containing cytochalasin B at 12.5 µM was added, and the cultures were incubated for a further 24 h (recovery period). Cytogenetic preparations from these cultures were obtained following standard procedures. The cytokinesis-block proliferation index (CBPI) was used to calculate cytotoxicity by the following formula:

cytotoxicity ) 100 - 100[(CBPIT - 1)/(CBPIC - 1)] where CBPI ) (no. mononucleate cells) + (2 × no. binucleate cells) + (3 × no. multinucleated cells)/total number of cells and CBPI was determined from at least 500 cells per culture. The cytotoxicity determined for each treatment level was used to select the highest dose level for scoring of micronuclei, which corresponded to a dose level that produced a cytotoxicity of approximately 60%. Four lower dose levels, evenly spaced, were also included in the scoring of micronuclei. The slides were randomly assigned code numbers by a person not subsequently involved in slide evaluation, and at least 1000 binucleated cells per cell cultures were scored to assess the frequency of micronucleated cells. The criteria for identifying micronuclei refers to ref 39. The numbers of binucleated cells with micronuclei in the control and treated cultures were compared using a χ-squared calculation. The test substance was considered positive when significant increases in the proportion of micronucleated cells over the concurrent controls occurred at one or more concentrations. However, this proportion at such data points had to exceed the normal range. If the increases fell within the range of values normally observed in the negative control cultures, the test compound was not classified as positive.

Chem. Res. Toxicol., Vol. 21, No. 6, 2008 1237 Table 1. MI Values Observed in V79 Cells and Human Lymphocytes in OTA-Treated Cultures in the Absence and Presence of Rat Liver S9 Metabolism treatment

Chromosomal Aberrations and SCEs. The results for MI are presented in Table 1 for V79 cells and human lymphocytes in the absence and presence of rat liver S9 metabolism and in Table 2 for V79 cells in the presence of rat kidney S9 metabolism. Analyses of chromosomal aberrations in V79 cells, in the presence of rat kidney S9 metabolism, are displayed in Table 3. Analyses of chromosomal aberrations and SCEs in V79 cells and human lymphocytes, following treatment of cultures with OTA in both the absence and the presence of rat liver S9 metabolism, are shown in Table 4. In V79 cells, OTA proved to be toxic at 2476.4 µM in both the absence and the presence of rat liver or kidney S9 metabolism where no metaphases were observed. At the immediate lower dose level (1149.0 µM), the MI values dropped to 52% of the relevant solvent control value in the absence of S9 metabolism and to 50 and 58% of the relevant solvent control values in the presence of liver or kidney S9 metabolism, respectively. At the successive lower dose levels, mitotic indices were slightly affected as compared to relevant solvent values.

S9 mix

untreated solvent (DMSO) OTA OTA OTA OTA OTA OTA OTA mitomycin-C untreated solvent (DMSO) OTA OTA OTA OTA OTA OTA OTA cyclophosphamide

V-79 cells 1% 2476.4 1149.0 532.4 247.6 114.9 53.2 24.8 0.9 + 1% + 2476.4 + 1149.0 + 532.4 + 247.6 + 114.9 + 53.2 + 24.8 + 23.6 +

untreated solvent (DMSO) OTA OTA OTA OTA OTA OTA OTA mitomycin-C untreated solvent (DMSO) OTA OTA OTA OTA OTA OTA OTA cyclophosphamide

human lymphocytes 1% 2476.4 1149.0 532.4 247.6 114.9 53.2 24.8 0.9 + 1% + 2476.4 + 1149.0 + 532.4 + 247.6 + 114.9 + 53.2 + 24.8 + 23.6 +

mean MI (%)

relative MI (%)

16.6 16.8 0.8 8.7 11.0 11.4 11.2 12.7 13.8 4.2 15.0 15.1 0.0 7.5 9.3 20.3 10.0 10.0 11.8 5.4

99 100 5 52 65 68 67 76 82 25 99 100 0 50 62 134 66 66 78 36

4.0 3.2 0.0 0.6 0.5 1.9 2.1 1.3 1.6 1.7 3.8 4.2 0.0 0.0 0.7 2.0 2.3 1.8 2.6 1.2

125 100 0 19 16 59 66 41 50 53 90 100 0 0 17 48 55 43 62 29

Table 2. MI Values Observed in V79 Cells in OTA-Treated Cultures in the Presence of Rat Kidney S9 Metabolism treatment

Results

dose level (µM)

untreated solvent (DMSO) OTA OTA OTA OTA OTA OTA OTA cyclophosphamide

dose level (µM) 1% 2476.4 1149.0 532.4 247.6 114.9 53.2 24.8 23.6

S9 mix

mean MI (%)

relative MI (%)

+ + + + + + + + + +

14.0 14.6 0.0 8.4 8.7 12.1 13.1 11.4 11.7 8.6

100 0 58 60 83 90 78 80 59

In human lymphocytes, OTA showed marked toxicity up to 532.4 µM, where MI dropped to values of 16 and 17% of the relevant solvent control in the absence and presence of rat liver S9 metabolism, respectively. At 247.6 µM, MI reached values 59 and 48% of the relevant solvent control in the absence and presence of rat liver S9 metabolism, respectively. As indicated in the previous section, the highest dose level to be selected for metaphase analysis should be a dose that produces a substantial reduction of MI as compared with the solvent control values, and ideally, the reduction should be approximately 50%. Treatments reducing the MI below 20% should not be used for

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Table 3. Frequencies of Chromosomal Aberrations and SCEs Observed in Cultured V79 Treated With OTA in the Presence of Rat Kidney S9 Metabolism total aberrations (%) treatment

dose-level (µM)

solvent (DMSO) OTA OTA OTA OTA cyclophosphamide

1% 114.9 247.6 532.4 1149.0 23.6

S9 mix + + + + + +

breaks

exchanges

aberrant cells (%)

mean SCEs per cell

endoreduplicated and polyploid cells (%)

4 2 4 4 5 8

0 1 0 0 0 4

4.0 3.0 4.0 4.0 5.0 12.0a

7.6 8.2 7.5 8.4 8.6 16.1d

0.0 0.0 7.0c 8.5b 13.0c 0.0

Statistically significant at p < 0.05 by Fisher’s exact test. b Statistically significant at p < 0.01 by Fisher’s exact test. c Statistically significant at p < 0.001 by Fisher’s exact test. d Statistically significant at p < 0.001 by Student’s t test. a

Table 4. Frequencies of Chromosomal Aberrations and SCEs Observed in Cultured V79 and Human Lymphocytes Treated With OTA in Both the Absence and the Presence of Rat Liver S9 Metabolism total aberrations (%) treatment

dose-level (µM)

S9 mix

breaks

exchange

aberrant cells (%)

mean SCEs per cell

endoreduplicated and polyploid cells (%)

V-79 cells solvent (DMSO) OTA OTA OTA OTA mitomycin-C solvent (DMSO) OTA OTA OTA OTA cyclophosphamide

1% 114.9 247.6 532.4 1149.0 0.9 1% 114.9 247.6 532.4 1149.0 23.6

+ + + + + +

2 3 3 4 3 20 3 3 3 7 6 15

0 1 0 1 1 12 0 1 0 0 2 10

2.0 4.0 3.0 5.0 4.0 30.0c 3.0 4.0 3.0 7.0 6.0 21.0c

6.6 6.2 7.3 6.5 9.0 37.0d 9.3 8.9 7.5 8.0 8.3 25.0d

0.0 4.0 16.0c 10.0b 13.0c 0.0 1.0 3.0 15.0c 9.0b 12.0c 0.0

human lymphocytes solvent (DMSO) OTA OTA OTA OTA mitomycin-C solvent (DMSO) OTA OTA OTA OTA cyclophosphamide

1% 24.8 53.2 114.9 247.6 0.9 1% 114.9 247.6 532.4 1149.0 23.6

+ + + + + +

0 3 0 5 1 11 3 3 3 7 6 16

0 0 0 0 0 9 0 1 0 0 2 10

0.0 3.0 0.0 5.0 1.0 20.0c 3.0 4.0 3.0 7.0 6.0 26.0c

3.4 3.7 3.6 3.4 4.2 24.0d 9.3 8.9 7.5 8.0 8.3 19.0d

0.0 9.0b 6.0a 10.0c 14.0c 0.0 0.0 4.0 8.0b 7.0b 13.0c 0.0

a Statistically significant at p < 0.05 by Fisher’s exact test. b Statistically significant at p < 0.01 by Fisher’s exact test. c Statistically significant at p < 0.001 by Fisher’s exact test. d Statistically significant at p < 0.001 by Student’s t test.

analyses of chromosomal aberrations and SCEs. On these bases, we selected the following dose levels for scoring: (i) V79 cells (absence and presence of S9 metabolism): 1149.0, 532.4, 247.6, and 114.9 µM; and (ii) human lymphocytes (absence and presence of S9 metabolism): 247.6, 114.9, 53.2, and 24.8 µM. Cytogenetic analyses showed that OTA did not induce any significant increase in chromosomal aberrations and SCE at any of the dose levels selected for scoring in V79 cell line and human lymphocytes in both the absence and the presence of rat liver S9 metabolism. Marked increases in the frequency of aberrant cells and SCEs were seen in the cultures treated with the positive control substances mitomycin-C and cyclophosphamide, indicating the correct functioning of the assay system. However, cytogenetic analyses revealed significant increases in the frequencies of both endoreduplicated and polyploid cells in V79 cells in the presence of kidney S9 metabolism (Table 3) and in V79 cells and human lymphocytes in both the absence and the presence of liver S9 metabolism (Table 4). Endoreduplicated and polyploid cells shown in Figure 1 are virtually absent or present at a very low incidence in both cell lines. Furthermore, a marked incidence of very condensed metaphases with abnormally separated

chromatids, as compared to untreated and positive control treated culture, was observed in the OTA-treated cultures (Figure 2). In Vitro Micronucleus Assay. The results for the CBPI to calculate cytotoxicity and the analyses of at least 1000 binucleated cells to evaluate the frequency of micronucleated cells in both the absence and the presence of S9 rat liver metabolism are presented in Tables 5 and 6, respectively. OTA proved to significantly affect the CBPI at 247.6 and 114.9 µM in both the absence and the presence of S9 metabolism where the calculated cytotoxicity was 100% or very close, respectively. At 53.2 µM, calculated cytotoxicity dropped to 67.2 and 69.3% in the absence and presence of S9 metabolism, respectively. In the in vitro micronucleus assay, cytotoxicity values approximately 60% calculated from the CBPI are considered to be ideal. On these bases, the dose levels selected for scoring of micronuclei were 53.2, 24.8, 11.5, and 5.3 µM in both the absence and the presence of S9 metabolism. Analyses of micronucleated binucleated cells showed that OTA did not induce any significant increase in micronuclei at any of the dose levels selected for scoring, in both the absence and the presence of rat liver S9 metabolism.

DNA NonreactiVe Mechanism of Ochratoxin A

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Figure 1. Endoreduplicated cells: (a) normal metaphase from a V79 cell line, (b) endoreduplicated cell from a V79 cell line treated with OTA at 247.6 µM, (c) normal metaphase from human lymphocytes, and (d) endoreduplicated cell from a culture of human lymphocytes treated with OTA at 247.6 µM.

Figure 2. (a) Normal metaphase from a V79 cell line. (b) Very condensed metaphase with abnormally separated chromatids from a V79 culture treated with OTA at 1149.0 µM.

Marked increases in the frequency of micronucleated cells were seen in the cultures treated with the positive control substances mitomycin-C and colchicine in the absence of S9 metabolism and cyclophosphamide in the presence of S9 metabolism, indicating the correct functioning of the assay system.

Discussion In the present paper, the results obtained clearly indicate that OTA does not show any clastogenic or aneugenic activity, based on the absence of induction of chromosomal aberrations, SCEs, and micronuclei in primary human lymphocytes and V-79 cells in both the absence and the presence of rat S9 metabolism in studies designed to comply with the experimental methods indicated in the OECD guidelines for the testing of chemicals no. 487 (draft, June 2004). This implies that OTA or possible metabolites do not covalently bind DNA through the formation

of DNA adducts or “bulky” adducts since structural chromosomal aberrations represent a very sensitive end point to detect chemical carcinogens with electrophilic substituents (40) and that clastogenicity and mutagenicity are characteristic of the majority of IARC group I human carcinogens. On the other hand, OTA was proved to induce endoreduplication, polyploidy, and very condensed metaphases with abnormally separated chromatids in V-79 cells and human lymphocytes in both the absence and the presence of S9 metabolism. Sister chromatids of metaphase chromosomes were clearly separated although in close proximity (Figure 2). Endoreduplication consists of two successive DNA synthetic periods without an intervening mitosis (Figure 1), leading to the formation of diplochromosomes in the following mitotic metaphase, thus generating tetraploid and polyploid cells. Polyploid cells are genetically unstable and lose chromosomes randomly to give aneuploidy (41–43). Aneuploidy is frequently found in human and rodent tumors characterized

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Table 5. CBPI Used to Calculate Cytotoxicity and Selection of Dose-Levels in the in Vitro Micronucleus Assay in Human Lymphocytes Treated With OTA in Both the Absence and the Presence of Rat Liver S9 Metabolisma treatment

dose-level (µM)

S9 mix

no. mononucleated

no. binucleated

no. multinucleated

CBPI

solvent (DMSO) OTA OTA OTA OTA OTA OTA OTA mitomycin-C colcemid solvent (DMSO) OTA OTA OTA OTA OTA OTA OTA cyclophosphamide

1% 2.5 5.3 11.5 24.8 53.2 114.9 247.6 1.5 0.1 1% 2.5 5.3 11.5 24.8 53.2 114.9 247.6 82.4

+ + + + + + + + +

2003 2006 2000 2012 2000 2000 2000 2000 2000 2000 2011 2014 2000 2000 2000 2002 2000 2000 2000

616 337 350 273 190 160 27 0 0 0 648 527 402 368 218 162 51 0 106

40 27 39 28 22 13 0 0 12 39 35 30 28 20 12 8 0 0 8

1.262 1.165 1.179 1.162 1.106 1.086 1.013 0.000 1.071 1.039 1.267 1.228 1.188 1.171 1.109 1.082 1.025 0.000 1.042

a

calculated cytotoxicity (%) 37 32 46 60 67 95a 100a 73 85 13 30 36 59 69 95a 100a 84

Highly cytotoxic.

Table 6. Distribution and Frequencies of Micronuclei in Human Lymphocytes Treated With OTA in Both the Absence and the Presence of Rat Liver S9 Metabolism MN distribution treatment

dose-level (µM)

S9 mix

binucleated scored

MN

0

1

2

3

solvent (DMSO) OTA OTA OTA OTA mitomycin-C colcemid solvent (DMSO) OTA OTA OTA OTA cyclophosphamide

1% 5.3 11.5 24.8 53.2 1.5 0.1 1% 5.3 11.5 24.8 53.2 82.4

+ + + + + +

1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000

1 2 6 5 4 20a 27a 3 1 3 3 5 32a

999 998 994 995 996 980 973 997 999 997 997 995 968

1 2 4 3 4 18 20 3 1 3 3 5 30

0 0 1 1 0 0 2 0 0 0 0 0 1

0 0 0 0 0 0 1 0 0 0 0 0 0

a

Statistically significant at p < 0.001 by χ-squared test.

by an elevated grade of invasivity (44–48). Endoreduplication, which is physiological in different organs of various organisms ranging from insects to plants and mammals (49–52), is also induced by different chemical and physical treatments including protein kinase inhibitors (53–55) through the induction of tetraploid cells at mitosis by interference with cell cycle check points and the regular development of mitotic events. Tetraploid cells thus result from mitotic exit in the absence of either chromosome segregation or cytokinesis through overriding of cell cycle check points or inhibition of microtubule assembly or inhibition of DNA topoisomerase II activity (43). It is interesting to note here that early pathological changes, observed in kidneys of rats administered OTA in vivo, include high levels of mitotically and multiple enlarged nuclei described as karyomegaly (37, 56–58). This was also seen by flow cytometry where the DNA content of giant nuclei, present in the medullary region of kidney of male Fischer F-344 rats administered OTA at 50 µg/kg body wt) for up to 2 years (tumor incidence 6/35) during experiments in a recent EU-funded program (QLKI-CT2001-011614), was greater than 4N.2 In a variety of human carcinomas, cells with tetraploid DNA contents arise as an early step in tumorigenesis and precede the formation of aneuploid cells (59). Examples of human tumors that develop in this way are esophageal adenocarcinoma (60, 61) and cervical carcinoma 2

Mosesso (2005) Personal communication.

(62). Tetraploidization also occurs as an intermediate step prior to aneuploidization and tumor formation in certain rodent model systems (63, 64). These findings further suggest a role for tetraploidization in tumor development from which clones with the capacity for tumor growth can develop (65), although recently, in experimental renal tumors induced in male Fisher rats in response to chronic OTA dietary exposure, it has been reported that all renal adenoma were diploid while all carcinomas were aneuploid (66). In vitro studies with mammalian cells showed that OTA severely affects cell cycle (30, 67). More specifically, in immortalized human kidney epithelial (IHKE) cells, OTA blocks mitosis at the metaphase-anaphase transition based on the modulation of the incidence of cells in prometaphase and cells at anaphase-telophase stages, and it simultaneously induces pro- and antiapoptotic signaling pathways resulting in mitotic cell death and premature exit from mitosis (67). Aberrant exit from mitosis appears to be supported by the activation of survival signals such as NF κB and ERK 1/2, which were shown to prevent apoptosis by promoting mitotic “slippage” (68, 69). Activation of NF κB appears to promote transition from G1 to S phases and allow re-entry of tetraploid cells into the cell cycle. This last condition is highly relevant for the induction of endoreduplication. After treatment with microtubule inhibitors (MTIs), cells defective in the CdK inhibitor p21 waf1/Cip1, which plays a regulatory role during the G1/S cell cycle check point, enter the S phase with a g 4N DNA content, a process

DNA NonreactiVe Mechanism of Ochratoxin A

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known as endoreduplication, which results in polyploidy, a condition that is highly unstable from the genomic point of view (70). Related to the findings of highly condensed metaphases with abnormally separated chromatids in V-79 cells and human lymphocytes following treatment with OTA, it is worth notice that cohesion of sister chromatids is maintained by cohesin, a multisubunit complex that groups-up the sister DNA strands by its ringlike structure. As cell progress through the prophase and prometaphase, cohesin is gradually removed under the control of Aurora and pololike kinases (Plk). At the metaphaseanaphase transition, cleavage of cohesin appears to require both activation of the protease “separase” through destruction of the inhibitory chaperone “securine” by the anaphase promoting complex (APC) and phosphorilation of cohesin subunits. This indicates that removal of cohesin from chromosomes could be modulated by different mitotic kinases like Cdk1, Aurora, or Plk (71, 72). More recently, cyclin B has also been shown to bind and inhibit separase, suggesting that untimely destruction of cyclin B may determine premature chromosome segregation. Collectively, these findings provide important evidence in support of an epigenetic mechanism of OTA carcinogenicity involving disruption of mitosis through interference with key regulators of chromosome separation and progression through mitosis such as Cdk1, cyclin B, Aurora, Plk, resulting in temporary blocked mitosis and subsequent premature exit from mitosis through “mitotic slippage” to generate endoreduplicated and polyploid cells accompanied by an increased risk of aneuploidy acquisition and subsequent tumor formation. Acknowledgment. Parts of this work were supported by the Fifth RTD Framework Program of the European Union, Project QLK1-2001-01614 “Mechanisms of Ochratoxin A Induced Carcinogenicity as a Basis for an Improved Risk Assessment” and by Italian M.U.R.S.T. (60%). The present paper is dedicated to Prof. A. T. Natarajan on the occasion of his 80th birthday.

Appendix 1

Treatment schedule used in the in vitro micronucleus assay.

Appendix 2a Protein Content and Aminopyrine Demethylase Activity Observed in S9 Tissue Homogenates in Sprague-Dawley Rats That Had Received Prior Treatment with Phenobarbital and Betanaphtoflavone to Induce High Levels of Xenobiotic-Metabolizing Enzymes

S9 batch

species/strain

organ

protein content (mg/mL)

2002/16 2003/01

rat/Sprague-Dawley rat/Sprague-Dawley

liver kidney

30.3 ( 1.25 15.7 ( 2.76

aminopyrine demethylase activity (µM/g liver/5 min, formaldehyde production) 3.48 ( 0.21 not performed

Appendix 2b S9 Tissue Fractions Were Prepared from the Livers and Kidneys of Five Young Male Sprague-Dawley Rats That Had Received Prior Treatment with Phenobarbital and Betanaphtoflavone to Induce High Levels of Xenobiotic-Metabolizing Enzymesa S9 batch

indirect mutagens

2002/16

negative control 2-aminoanthracene (1 µg/plate) benzo(a)pyrene (2.5 µg/plate) negative control 2-aminoanthracene (1 µg/plate) benzo(a)pyrene (2.5 µg/plate)

2003/01

induced revertants 160 ( 8 1376 ( 24 516( 35 182 ( 12 1508 ( 145 213( 17

a The efficacy of the S9 tissue fractions was previously checked in an Ames test with the indirect mutagens 2-aminoanthracene and benzo(a)pyrene, using S. typhimurium tester strain TA100.

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