| The micronucleus test is a cytogenetic method with clear endpoints of detection: both chromosome loss and breakage. It is frequently used to study the genetic toxicity of xenobiotics, and its mechanisms when combined with centromere immunofluorescence technique. The in vitro micronucleus test has gradually become an important screening test of unknown chemicals for their potential genotoxicity, by reason of the animal welfare ethics requirement and the "3R" (Refinement, Reduction, Replacement) principle of animal experiment. Currently, the in vitro micronucleus test models mainly include experiment employing human lymphocytes and that in mammalian cell lines; the former is advantageous for its use of human cells, while the latter which is performed in immortal mammalian cell lines is with rapid cell growth, stable karyotype, and good reproducibility. The standard procedures of micronucleus test in immortalized mammalian cell lines include the following exposure/recovery schedules:exposure of test chemicals to the test cells for a short time (≤ 0.5 cell cycle), and then a long time (≥ 1.5 cell cycles) period for recovery (freedom of test chemicals). If the result is negative or equivocal, a second experiment with the following procedure is required:a long exposure time (≥ 2 cell cycles) without recovery before the harvest of cells. This set of experiment procedures help minimizing false negative results. It has been reported that clastogens and aneugens could be detected with these two experimental procedures, respectively. Numerous important environmental mutagens and carcinogens can exert genotoxic action only after metabolic activation by specific biotransformation enzymes. Therefore, using metabolic activating systems in the in vitro genotoxicity assays is a common strategy to prevent from false negative results due to no or insufficient activities of metabolic enzymes expressed in the standard test cells. The classic external bioactivating system, i.e., the Aroclor 1254-induced rat liver S9 fraction supplemented with an NADPH-generating system (S9 mix), has been applied in the Ames test and other in vitro genotoxicity assays for detection of the mutagenicity of some chemicals metabolically dependent on a few cytochrome P450 (CYP) enzymes. However, this system may not be optimal for the detection of some indirect mutagens due to various reasons. For example, CYP1B1 and CYP2E1 and a large number of non-CYPs biotransformation enzymes required for metabolic activation of chemicals are absent from S9 mix. In addition, the exposure time of S9 mix may be limited in the in vitro micronucleus test due to the instability of enzymes and lipid peroxidation. Therefore, expression of human xenobiotic-metabolizing enzymes in cell lines commonly used in genotoxicity assays aids greatly in the detection of enzyme-dependent genotoxicants.To confirm the preference of differential exposure/recovery schedules in the detection of clastogens and aneugens, as proposed in earlier reports. The first part of this thesis is to detect micronucleus cells by known clastogens (mitomycin C and bleomycin) and aneugens (colchicine and vinblastine) in Chinese hamster lung fibroblast (V79) cells with different exposure/recovery schedules (0h/24h,3h/21h, 6h/18h,12h/12h,18h/6h and 24h/0h). However, the process of metabolic activation of indirect mutagen in cells expressing biotransformation enzymes may encounter enzyme protein saturation and be time-requiring.Whether it affects the specific relationship between a preferred exposure/recovery schedule and sensitive induction of micronuclei has not been reported. The second part of this thesis is to study genetic toxicity mechanism of xenobiotic substances requiring metabolic activation through a relationship between exposure/recovery and induction of micronuclei established in the first part of this thesis. Recently we have identified that 1-methylpyrene (1-MP) is activated sequentially by human CYP2E1 and human SULT1A1; meanwhile 1-hydroxymethylpyrene (1-HMP) is activated by human SULT1A1 (both compounds are non-genotoxic without metabolic activation). We also have observed activation of hydroquinone (HQ), catechol (CAT) and 1,2,4-trihydroxybenzene (THB) by human CYP2E1 to more genotoxic metabolites. The second part of this thesis is to investigate the micronuclei inducing effects the above chemicals in a V79-derived cell line expressing both human CYP2E1 and human SULT1A1 (V79-hCYP2E1-hSULT1A1) with different exposure/recovery schedule. Also in this part, the effects of the above chemicals on the frequency of mitotic cells were determined, for an estimation of the presence of mitotic arrest, which indicates disruption of microtubules and a potential aneugenic effect.Polychlorinated biphenyls (PCBs) are a group of persistent organic pollutants which are still present in large quantities in the global environment, and also accumulated to varied degrees in high-exposure human populations. In 2013, PCBs have been classified by the International Agency for Research on Cancer (IARC) as human (group 1) carcinogens. CYP2E1 is an important biotransformation enzymes mainly expressed in the liver, which can catalyze the metabolism of numerous small molecular compounds, including benzene, phenol, nitrosamines, and some short chain halogenated aliphatic hydrocarbons. Through a preliminary study utilizing a series of PCB compounds in the micronucleus test and the Hprt mutagenicity assay, we have observed that lower chlorinated PCBs could be activated by human CYP2E1 to genotoxic metabolites, a metabolic activating mechanism much stronger than those by several other CYP enzymes. Mouse is a species of commonly used animals in toxicological studies, however, there has been no report about the metabolic activation of PCBs by mouse CYP2E1. In order to investigate the similarity of difference between CYP2E1 of the human and mouse species in activating PCBs to mutagenic metabolites, in the third part of the thesis, we used four PCBs, i.e., PCB 5, PCB 18, PCB 20 and PCB 28, which induce micronuclei in a decreasing order (as observed in the preliminary study), and N-nitrosodimethylamine (a strong mutagen activated by CYP2E1) as a reference compound, in the mutagenicity assay at the Hprt locus in both a V79-derived cell line expressing mouse CYP2E1 (V79-mCYP2E1) and V79-hCYP2E1-hSULT1A1 cells. The capabilities of mouse and human CYP2E1 in activating individual PCB compounds to mutagens were then compared, which may provide a basis for choosing an animal species suitable for toxicological studies in vivo.Objective1. To confirm whether varied exposure/recovery procedure preferred for induction of micronuclei in mammalian cell lines by different xenobiotics represents distinct mechanisms of genotoxicity:clastogenesis and aneugenesis.2. To investigate the induction of micronuclei by several xenobiotics requiring metabolic activation in a mammalian cell line genetically engineered for expression of biotransformation enzymes under various exposure/recovery schedules, to ascertain whether the time-requiring metabolism has an influence on the dynamics of micronuclei formation, and the possible mechanisms for the genotoxicity of these indirect genotoxicants.3. To investigate the roles of human and mouse CYP2E1 in the induction of gene mutations at the Hprt locus by NDMA and four PCBs, and thus the similarity or difference of these two enzymes in activating PCBs to mutagens.Methods1. Cytotoxicity testThe cytotoxicity of each chemical was determined in the cell lines using CCK-8 assay (disulfonated tetrazolium salt serving as a chromogenic indicator for NADH and cell viability). The V79 cells and V79-hCYP2E1-hSULT1A1 cells were exposed to test compounds with a set of exposure/recovery schedules as Oh/24h (negative control),3h/21h,6h/18h,12h/12h,18h/6h, and 24 h/0 h. Finally the optical density of each sample at 450 nm (OD450) was measured on a microplate reader. The cell growth/viability level was indicated by a comparison of OD450 values between each exposure group and the control group.2. Micronucleus testThe micronucleus test is one of the commonly used assays for detecting transmittable cytogenetic changes. V79 cells and V79-hCYP2El-hSULT1A1 cells were inoculated separately, at 24h, cells were exposed to test compounds with varied exposure/recovery schedules as mentioned in CCK-8 assay. Two concentrations of each test chemical with no or mild cytotoxicity accompanied by obvious micronuclei induction were used. For the test compounds metabolized by SULT1A1 (HQ, CAT and THB), inhibition of the expressed SULT1A1 in V79-hCYP2E1-hSULT1A1 cells was attained by the presence of PCP, at 22h-48h. Cells in each culture were harvested by trypsinization, subject to hypotonic treatment, fixed in methanol:acetic acid (3:1) mixture, dropped onto glass slides, and finally stained with Giemsa stain. In each treatment group duplicate determinations were designed. Under microscopy with the oil immersion lens (magnification of 1000),1000 randomly encountered cells (with integrate cell structure) from each culture were scored for the frequency of micronucleated cells, each containing one or more micronuclei in the cytoplasm (separated from the main nucleus), by an experienced experimenter.3. Determination of the frequency of mitotic cellsA prominent manifestation of cellular spindle damage is mitotic arrest, manifested by increased mitotic frequency without enhanced cell growth. A 4.5cm2 round glass slide was placed onto each well (9.6cm2) of a series of 6-well culture boards; then, cells of each line were inoculated onto each well. At 24h, cells in each culture were treated with each test chemical at two different concentrations (the same as in the micronucleus test). For HQ, CAT and THB inhibition of the expressed SULT1A1 was attained by the presence of PCP (10μM) at 22h-48h. At 48h, the culture medium in each culture was removed, and PBS was used to clean each well for twice. Immediately after each well got dry, cells were fixed with 1mL of methanol: acetic acid (3:1), and finally stained with Giemsa stain. The glass slide in each culture was taken out for microscopic observation of mitotic index; under 1000-fold magnification,1000 randomly encountered and structurally integrate cells were scored for a frequency of mitotic cells.4. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western BlotSDS-PAGE and Western Blot are a common method of electrophoresis separation and quantitative analysis of protein. The major steps of the experiments was as follows:preparation of protein samples, SDS-PAGE (the protein sample amount was 50μg/20μL), electrotransfer, antibody blocking, antibody incubation, TBST washing, electrochemical chromogenic reaction and analysis.5. Mutagenicity assayForward mutations at the Hprt locus, demonstrated by the acquisition of resistance to 6-thioguanine, was used as a marker of gene mutations. Prior to the mutagenicity assay, pre-existing Hprt mutants in V79-hCYP2E1-hSULT1A1 cells were removed by culturing for three passages in HAT medium containing aminopterin, an inhibitor of dihydrofolate reductase prohibiting de novo synthesis of purine nucleotides.1.0 x 106 cells were inoculated ontoeach 182cm2 culture flask in 25mL medium. At 24h, cells were exposed to test compounds at varying concentrations. Each culture was continued for another 24h, and then changed with fresh medium. At 4d, the cells were detached by treatment with trypsin (cell numbers expressed as a percent of the numbers in the control group were used as a measure of the cytotoxicity), subcultured for 3 days, and then subcultured again using medium supplemented with 6-thioguanine (7μg/mL) for the selection of mutants (106 cells per 137cm2 Petri dish,4 dishes) and normal medium for determining the colony-forming efficiency (100 cells per 9.6cm2 Petri dish,3 dishes). After 8d, the cultures were fixed and stained, the colonies were counted and mutant frequencies were calculated from the averaged values for each initial culture.6. Statistical analysisANOVA was performed to analyze the results of cytotoxicity assay with each test chemical, with the relative optical density (in percentage) in each treatment compared with that in the control group. The micronucleus and mitotic index assays were analyzed with the x2 test after combining the data from duplicate assays. The mutant frequency in each treatment was compared with that in the control by using the exact probability (Fisher) method.Results1. Induction of micronuclei by MMC and BLM in V79 cells displayed a biphasic feature:an exposure duration-dependent increase in micronuclei formation up to 12h/ 12h treatment, followed by a reduced effect with extended exposure durations. Differently, COL and VBL induced micronuclei in an exposure length-dependent manner consistently, reaching the highest activity at the 24h/0h schedule. Additionally, while MMC and BLM had no effect on the frequency of mitotic cells, COL and VBL significantly induced a mitotic arrest in V79 cells. Correspondingly, COL and VBL mildly or moderately prohibited cell growth/viability, but MMC an BLM did not show a substantial effect. The above results provided an appropriate reference for future studies regarding the adoption of an appropriate exposure/ recovery schedule in micronucleus test in cells expressing biotransformation enzymes.2. Similar to the induction of micronuclei by COL and VBL in V79 cells,1-MP and 1-HMP elevated the frequency of micronucleated in V79-hCYP2E1-hSULT1A1 cells along with the increase in exposure duration, peaked at 18h/6h. The induction of micronuclei by the hydroxylated metabolites of benzene in V79-hCYP2E1-hSULT1A1 cells resembled that by the known clastogens in V79 cells: the induction of micronuclei by CAT and THB peaked at the 6h/18h schedule and e HQ peaked at schedules with somewhat longer exposure. In addition,1-MP and 1-HMP showed mild to moderate cytotoxicity in V79-hCYP2E1-hSULT1A1 cells in an exposure duration-dependent manner; however, HQ, CAT and THB did not show significant cytotoxicity in the cells. Furthermore,1-MP and 1-HMP apparently induced a mitotic arrest in V79-hCYP2E1-hSULT1A1 cells, while CAT, THB and HQ did not have a substantial effect. Under varying exposure/recovery schedules, the induction of micronuclei and mitotic arrest in V79-hCYP2E1-hSULT1A1 cells by each test compound was greater at the high concentration than at low concentration.3. Western-blotting results showed that the relative levels of CYP2E1 expression in V79-mCYP2E1 and V79-hCYP2E1-hSULT1A1 cells increased sequentially. All the five test compounds did not induce gene mutations in V79 cells, however, NDMA induced strong gene mutations in both V79-mCYP2E1 and V79-hCYP2E1-hSULT1A1cells, apparently in a concentration-dependent manner. PCB18 and PCB5 were both strongly mutagenic in V79-hCYP2E1-hSULT1A1 cells, but they were only weakly positive in V79-mCYP2E1 cells. Both PCB 20 and PCB 28 were marginally mutagenic in V79-hCYP2El-hSULTlAl and V79-mCYP2E1 cells.Conclusion1. In accordance to previous reports, under varying exposure/recovery schedules, clastogens and aneugens demonstrate distinct difference in the induction of micronuclei in cultured mammalian cells, reaching maximal effects under 12h/12h and 24h/Oh schedule, respectively. In the in vitro micronucleus test in mammalian cell lines, a short exposure combined with long recovery is favorable for detection of clastogens, while a long exposure and no recovery may sensitively detect aneugens.2. According to the above relationship between the induction of micronuclei by clastogens and aneugens and exposure/recovery schedules, meanwhile, together with the differences in the induction of micronuclei and mitotic arrest in V79-hCYP2E1-hSULT1A1 by the individual test compounds, it is concluded that the two alkylated pyrene compounds (1-MP and 1-HMP) may be activated by CYP2E1 and/or SULT1A1 to to a microtubule-damaging and and aneugenic metabolite. On the contrary, among the hydroxylated metabolites of benzene, CAT and THB are probably clastogens, and HQ may possess both clastogenic and aneugenic activities. Although metabolic activation requires time, the induction of micronuclei by the indirect mutagens applied in this study was not delayed, as compared with that by the direct mutagens, therefore, the standard procedure of the micronucleus test in cell lines may still applicable to a test with target cells expressing metabolic enzymes, at least in the cell model employed in the present study.3. The test PCB compounds demonstrate great variations in the potency and efficacy in the induction of gene mutations in V79-derived cells expressing CYP2E1 of different species:human and mouse, particularly in the case of PCB 18 and PCB 5. This cannot be explained by the varied levels of CYP2E1 protein in the test cell lines, and instead the difference in the enzyme proteins of two species should be primarily contributive. Therefore, use of mouse as an experimental model in vivo to analyze the genotoxicity of PCBs is not suitable for an essential metabolic activation, and the suitability of other animal models (such as rat, rabbit) for this study purpose still needs further investigation. |
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