Exogenous Metabolic Activation Research Articles

Ethidium bromide and SYBR Green I enhance the genotoxicity of UV-irradiation and chemical mutagens in E. coli

Ethidium bromide (EtBr) and SYBR Green I are nucleic acid gel stains and used commonly in combination with UV-illumination. EtBr preferentially induces frameshift mutations but only in the presence of an exogenous metabolic activation system, while SYBR Green I is a very weak mutagen that induces frameshift mutations. We found that EtBr and SYBR Green I, without an added metabolic activation system, strongly potentiated the base-substitution mutations induced by UV-irradiation in E. coli B/r WP2 cells. Each DNA stain alone showed no mutagenicity to the strain. Base-substitutions induced by 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5 H)-furanone (MX) and 4-nitroquinoline-1-oxide were similarly potentiated by EtBr and SYBR Green I. SYBR Green I had a much greater effect. No enhancing effects were observed on mutations induced by mitomycin C, cisplatin, transplatin, cumene hydroperoxide, base analogs, and alkylating agents. Another DNA stain, acridine orange, showed similar enhancing effects on UV- and MX-mutagenicity, but no effect was observed for 4′,6-diamidino-2-phenylindole (DAPI). UV- and MX-induced mutations in E. coli WP2s ( uvrA), which is defective in nucleotide excision repair activity, were not potentiated by the addition of EtBr, SYBR Green I, or acridine orange. Those nucleic acid stains might inhibit the nucleotide excision repair of DNA damaged by UV and MX treatment.

A comparison of aroclor 1254-induced and uninduced rat liver microsomes to human liver microsomes in phenytoin O-deethylation, coumarin 7-hydroxylation, tolbutamide 4-hydroxylation, S-mephenytoin 4′-hydroxylation, chloroxazone 6-hydroxylation and testosterone 6β-hydroxylation

Aroclor 1254-induced rat liver homogenate supernatant (liver S-9) is routinely used as an exogenous metabolic activation system for the evaluation of mutagenicity of xenobiotics. The purpose of this study is to evaluate whether results obtained with Aroclor 1254-induced liver microsomes would be relevant to human. Aroclor 1254-induced and uninduced rat liver microsomes were compared to human liver microsomes in the metabolism of substrates which are known to be selectively metabolized by the major human cytochrome P450 (CYP) isoforms. The activities studied and the major CYP isoforms involved were as follows: phenacetin O-deethylation (CYP1A2); coumarin 7-hydroxylation, (CYP2A6); tolbutamide 4-hydroxylation (CYP2C9), S-mephenytoin 4′-hydroxylation (CYP2C19); dextromethorphan O-demethylation (CYP2D6); chloroxazone 6-hydroxylation (CYP2E1); and testosterone 6β-hydroxylation (CYP3A4). We found that both induced and uninduced rat liver microsomes were active in all the pathways studied with the exception of coumarin 7-hydroxylation. Coumarin 7-hydroxylation was observed with human liver microsomes but not the rat liver microsomes. Aroclor-1254 was found to induce all activities measured, with the exception of coumarin 7-hydroxylation. Dextromethorphan O-deethylation activity was higher in the rat liver microsomes than the human liver microsomes. Testosterone 6β-hydroxylation activity was found to be similar between the human liver microsomes and the induced rat liver microsomes. Our results suggest that experimental data obtained with Aroclor 1254-induced rat liver microsomes may not always be relevant to human.

Is ethanol genotoxic? A review of the published data.

A great many studies have been carried out on the toxicology of ethanol, the majority in the context of the effects of the consumption of alcohol in beverages. Published information relevant to the assessment of the possible genotoxic potential of ethanol has been reviewed and evaluated in terms of the safety of ethanol as an industrial chemical, rather than as a component of beverages. The available data on ethanol from standard genotoxicity test methods are incomplete. There is clear evidence that ethanol is not a bacterial or mammalian cell mutagen but in vitro assays for chromosome aberration, although mostly negative, have generally not included exogenous metabolic activation. Evidence from the use of ethanol as a vehicle control suggests that it is not mutagenic or clastogenic in vitro. Reported tests for chromosome aberration induction in vivo are all negative and only a minority of micronucleus tests are positive. Conflicting results have been reported for the dominant lethal assay, although an inter-laboratory study performed to OECD guidelines was negative. There is some evidence that ethanol induces SCE in vivo and can also act as an aneugen at high doses. Many in vivo studies were designed to model alcoholism and used very high doses, sometimes for long periods. Outcomes may have been affected by disturbances of metabolism giving rise to secondary effects. It is concluded that there is no significant evidence that ethanol is a genotoxic hazard according to the criteria normally applied for the purpose of classification and labelling of industrial chemicals. Some degree of genotoxicity may result from excessive alcohol drinking, but this is not considered relevant to any conceivable exposure obtainable by either inhalation or dermal exposure in the workplace.

OPTIMIZATION OF AN EXOGENOUS METABOLIC ACTIVATION SYSTEM FOR FETAX. I. POST-ISOLATION RAT LIVER MICROSOME MIXTURES

The developmental toxicity of cyclophosphamide, coumarin, 2-acetylaminofluorine (2-AAF), and trichloroethylene (TCE) was assessed with Frog Embryo Teratogenesis Assay: Xenopus (FETAX). Late Xenopus laevis blastulae were exposed to each test material for 96-h in two separate static-renewal tests with and without the presence of five differently induced exogenous metabolic activation systems (MAS). The MAS consisted of Aroclor 1254- (Aroclor 1254 MAS), isoniazid- (INH MAS), phenobarbital- (PB MAS), or β-naphthoflavone- (β-NF MAS), or a post-isolation mixture (mixed MAS) of INH-, PB-, and β-NF-induced rat liver microsomes. Addition of the Aroclor 1254 MAS bioactivated cyclophosphamide, coumarin, 2-AAF, but not TCE. Addition of the PB MAS bioactivated cyclophosphamide, weakly bioactivated coumarin and 2-AAF, but had no effect on TCE developmental toxicity. The β-NF MAS bioactivated coumarin and 2-AAF, weakly bioactivated cyclophosphamide, but did not alter the developmental toxicity of TCE. Addition of the INH-induced MAS only bioactivated TCE, whereas the post-isolation mixed MAS bioactivated each test material. Based on LC50 and EC50 (malformation)values, embryo growth, and types and severity of induced malformations, each test material was developmentally toxic. Use of post-microsome isolation mixtures from differentially induced rat livers increased the efficacy of the exogenous MAS routinely used by FETAX.

Cytological and cytogenetic effects of the fluoroquinolone ofloxacin on human periodontal ligament fibroblasts.

The fluoroquinolone ofloxacin (OFLX) is one of the candidates of antibacterial agents to be topically used against periodontitis. To estimate the maximum concentration of OFLX which exerts little or no adverse effect on the periodontal ligament, cytological and cytogenetic effects of OFLX on human periodontal ligament fibroblasts (Pel cells) were examined. Treatment of Pel cells with < or =0.3 mM OFLX for 24 or 48 h had little inhibitory effect on cellular growth and survival. DNA, RNA and protein syntheses in Pel cells did not decrease in response to treatment with < or =0.3 mM OFLX. The constitutive level of alkaline phosphatase mRNA was retained in cells treated with < or = 0.03 mM OFLX for 24 or 48 h. The level of type I procollagen mRNA was not affected by treatment with < or = 0.003 mM OFLX for 24 or 48 h. Cytogenetic effects of OFLX were evaluated by the ability of OFLX to induce chromosome aberrations in Pel cells. Treatment with OFLX at 0.3-3.0 mM for 6, 24, or 48 h failed to induce chromosome aberrations in Pel cells. The failure of OFLX to induce chromosome aberrations was seen even in the presence of exogenous metabolic activation using a 5% rat liver post-mitochondrial supernatant mixture. These results indicate that treatment of Pel cells with < or =0.003 mM OFLX has few or no adverse effects on the cytological and cytogenetic endpoints examined, suggesting that there would be little adverse effect on growth and differentiation of the periodontal ligament, as well as little cytogenetic activity, if OFLX were to be topically administered to the gingival crevice at the minimal inhibitory concentration (MIC90) against periodontopathic bacteria (< or = 0.0027 mM). It is important to note, however, that extrapolation of these findings to in vivo conditions has yet to be undertaken.

Cytogenetic effect of the anticancer drug epirubicin on Chinese hamster cell line in vitro

The present study demonstrated the cytogenetic effect of the anticancer drug epirubicin on cultures of Chinese hamster cell line in vitro. The cultures were exposed to the drug for 24 h at three final concentrations; 10, 20 and 40 μg/ml. All treatments were carried out in the absence of any exogenous metabolic activation system. The different types of structural chromosomal aberrations, including gaps, breaks, deletions and fragments were increased in epirubicin-treated cultures. This increase was dose dependent where there was a positive correlation between increased drug concentration and induction of structural chromosomal aberrations. Also, the numerical chromosomal aberrations, including hypodiploidy and hyperdiploidy, were increased significantly in epirubicin-treated cultures. Like structural aberrations, the increase of numerical chromosomal aberrations was also dose-dependent. The frequency of sister chromatid exchanges in cultures treated with epirubicin increased significantly and this increase was dose-dependent. On the other hand, the epirubicin significantly decreased the mitotic index in treated cultures of Chinese hamster cell line.

Evaluation of acute toxicity and genotoxicity of liquid products from pyrolysis of Eucalyptus grandis wood.

Slow pyrolysis of Eucalyptus grandis wood was performed in an oven laboratory, and smoke was trapped and condensed to yield liquid products. Polycyclic aromatic hydrocarbons (PAHs) and phenolic fractions were isolated from the former liquid products using adsorption column chromatography (ACC) and identified by GC/MS. Concentrations of PAH and phenolic fractions in total pyrolysis liquids were respectively 48.9 microg/g and 8.59% (w/w). Acute toxicity of total samples of pyrolysis liquids and the phenolic fraction was evaluated by means of two bioassays, namely, 24-h immobilization bioassay with Daphnia magna and Microtox bioassays, the latter employing the luminescent bacteria Photobacterium phosphoreum. Total pyrolysis liquids and the PAH fraction were evaluated for genotoxicity by the Microtox bioassay conducted using rehydrated freeze-dried dark mutant of the luminescent bacteria Vibrio fisheri strain M169. Total pyrolysis liquids and the phenolic fraction, respectively, in concentrations of 170 and 68 mg/L were able to immobilize 50% (EC(50)) of the D. magna population following 24-h exposure. Concentrations of 19 and 6 mg/L, respectively, for total pyrolysis liquids and phenolic fraction were the effective concentrations that resulted in a 50% (EC(50)) reduction in light produced by bacteria in the Microtox bioassay. Accordingly, the Microtox bioassay was more sensitive to toxic effects of both kind of samples than the D. magna bioassay, particularly for the phenolic fraction. Regarding to the genotoxicity evaluation, the results achieved by Microtox bioassay showed that total pyrolysis liquids had no genotoxic effects with and without exogenous metabolic activation using rat liver homogenate (S9). However, the PAH fraction showed toxic effects with rat liver activation and had a dose-response number (DRN) equal to 1.6, being in this way suspected genotoxic. The lowest detected concentration (LDC) of the PAH fraction able to cause genotoxic effects was 375 microg/L.

Use of the Zebra Mussel (Dreissena polymorpha) as a Bioindicator for Aromatic Hydrocarbons in Hamilton Harbour

Abstract Three methods for the extraction of polycyclic aromatic hydrocarbons (PAHs) from zebra mussels (Dreissena polymorpha) sampled from Hamilton Harbour were compared. Replicate freeze-dried mussel tissue samples were extracted using acid digestion, tissue homogenization (mechanical extraction) and ultra-sonication. Each extract was submitted to a cleanup procedure (alumina chro-matography and Sephadex LH20 gel chromatography), followed by analysis using gas chromatography-mass spectrometry (GC-MS). The three extraction methods were equally efficient, based on a statistical comparison of mean concentrations of individual PAHs. Mussel extracts, when subjected to bioassays with Salmonella typhimurium strain YG1029 (TA100-like) in the presence of an exogenous metabolic activation system (S9), exhibited significant mutagenic responses; these responses varied with the PAH content of the mussel extracts. Sources of PAHs in mussel extracts were determined by examining the profiles of sulfur-containing polycyclic aromatic compounds (thia-arenes). Comparison of the ratios of certain thia-arenes with ratios in source samples enabled identification of vehicular emissions and coal tar-contaminated sediment as two sources of PAH contamination in Hamilton Harbour.

Cloning and expression of rat CYP2E1 in Saccharomyces cerevisiae: detection of genotoxicity of N-alkylformamides.

A cDNA coding for rat cytochrome P450 2E1 was cloned into the multicopy vector pYeDP60 and expressed in haploid RSY6 and diploid RS112 yeast strains of Saccharomyces cerevisiae under control of the GAL10-CYC1 promoter. Spectral and catalytic properties of the expressed 2E1 were examined in whole cells or microsomes of both strains. The level of CYP2E1 obtained in RS112 (200 pmol/mg microsomal protein) was the highest among CYP2E1 produced in the various expression systems. The monooxygenase activity in the microsomes of both strains, measured as aniline hydroxylase, was found comparable to that of control rat hepatic microsomes. In a reconstituted system in the presence of exogenous rat P450 reductase, their activity increased about 10-fold. When exposed to the carcinogen NDMA, a known 2E1 substrate, the recombination frequency determined in the 2E1-expressing RS112 cells was enhanced, in a dose-dependent manner, up to 20-fold. The exposure of the same cells to the hepatotoxic solvents, N-methyl- and N-ethylformamide, resulted in an induction of recombination frequency, which was not observed in the void plasmid containing RS112 cells in the presence of S9 hepatic fractions from pyrazole-induced rats, as a specific exogenous metabolic activation system. These results demonstrate that the 2E1-expressing cells metabolize the two N-alkylformamides to genotoxic intermediates and, therefore, they provide an useful tool to study the bioactivation mechanism of potential P450 2E1 substrates.

Assessment of genotoxicity of dental antiseptics: ability of phenol, guaiacol, p-phenolsulfonic acid, sodium hypochlorite, p-chlorophenol, m-cresol or formaldehyde to induce unscheduled DNA synthesis in cultured Syrian hamster embryo cells.

To assess the genotoxicity of seven dental antiseptics, the ability of these agents to induce unscheduled DNA synthesis (UDS) was examined using Syrian hamster embryo (SHE) cells. Treatment of SHE cells with phenol or formaldehyde induced UDS in a concentration-dependent manner as determined by direct scintillation counting of [3H]thymidine incorporated into DNA during repair synthesis. Guaiacol or m-cresol induced UDS only in the presence of exogenous metabolic activation. p-Phenolsulfonic acid, sodium hypochlorite and p-chlorophenol failed to induce UDS in the presence or absence of exogenous metabolic activation. Our results suggest that phenol, guaiacol, m-cresol and formaldehyde are genotoxic to mammalian cells.

Evaluation of the developmental toxicity of thalidomide using frog embryo teratogenesis assay-xenopus (FETAX): biotransformation and detoxification.

The developmental toxicity of thalidomide was evaluated using FETAX (Frog Embryo Teratogenesis Assay - Xenopus). Young X. Laevis embryos were exposed to this compound in each of two concentration-response experiments with and without differently induced exogenous metabolic activation systems (MASs) and/or inhibited MASs. Young male Sprague-Dawley rats were treated with either isoniazid or Aroclor 1254 to induce cytochrome P-450. Several of the rats were subsequently treated with diethyl maleate (DM) to deplete glutathione reserves. Specific aliquots of rat liver microsomes were treated with 3-amino-1,2,4-triazole (ATZ) or alpha-napthoflavone (alpha-N) to selectively inhibit P-450 activity. Bioactivation was indicated by increased developmental toxicity observed in MAS tests. Results obtained indicated that thalidomide was predominantly activated by P-450 isozyne CYP2E1, although weak cross-specificity between CYP1A1/A2 may have existed. Detoxification pathways for thalidomide were investigated by treatment of the MAS with cyclohexene oxide (CHO) and DM to inhibit the epoxide hydrolase and glutathione conjugation pathways, respectively. Results indicated that epoxide hydrolase was primarily responsible for the detoxification of bioactivated thalidomide. Teratogenesis Carcinog. Mutagen. 20:35-47, 2000.

Phase III interlaboratory study of FETAX. Part 3. FETAX validation using 12 compounds with and without an exogenous metabolic activation system.

FETAX (Frog Embryo Teratogenesis Assay-Xenopus) is a 96-h whole-embryo developmental toxicity screening assay that can be used in ecotoxicology and in detecting mammalian developmental toxicants when an in vitro metabolic activation system is employed. A standardized American Society for Testing and Materials (ASTM) guide for the conduct of FETAX has been published, along with a companion atlas that helps in embryo staging and in identifying malformations. As part of the ASTM process, an interlaboratory validation study was undertaken to evaluate the repeatability and reliability of FETAX and to evaluate the potential teratogenic hazard of 12 compounds. Three different laboratories participated in the study. All three participating laboratories had extensive experience with the assay. FETAX intralaboratory and interlaboratory variability, as judged by coefficients of variation, were very low. Potential teratogenic hazard was evaluated using two major criteria from FETAX experiments employing metabolic activation systems (MAS). These were the teratogenic index TI (TI = 96-h lc(50)/96-h ec(50) (malformation)) and the minimum concentration that inhibits growth (MCIG). A compound was considered teratogenic by this criterion when the MCIG was significantly different from controls at concentrations below the 30% level of the MAS 96-h lc(50). Based on the results of this and other studies, a decision table was constructed in order to evaluate additional studies. Severity of malformations caused, especially near the MAS 96-h ec(50) (malformation), were also evaluated. Four compounds were non-teratogenic but two compounds were clearly teratogenic. The remaining six compounds were ranked as equivocal teratogens. The results were discussed in light of the difficulty of producing an adequate decision table. FETAX proved to yield repeatable and reliable data as long as care was taken during range-finding and technicians were adequately trained. The MAS was essential in using FETAX to predict developmental hazard in mammals, and still requires further development.

Phase III interlaboratory study of FETAX part 3. FETAX validation using 12 compounds with and without an exogenous metabolic activation system

Phase III interlaboratory study of FETAX part 3. FETAX validation using 12 compounds with and without an exogenous metabolic activation system

Genotoxicity testing of six insecticides in two crosses of the Drosophila wing spot test

Among the great variety of genotoxicity assays available, the wing spot test in Drosophila melanogaster has some characteristics that make it very suited for the screening of genotoxic activity, i.e., it is an easy and inexpensive assay using a eukaryotic organism in vivo. One of the most interesting characteristics of the assay is its capacity to detect genotoxic activity of promutagens without the necessity of an exogenous metabolic activation system. In this paper we present results obtained with a recently developed high bioactivation cross of the wing spot test (NORR cross). The positive results obtained with the five well-known procarcinogens 7,12-dimethylbenz[ a]anthracene, N-nitrosopyrrolidine, p-dimethylaminoazobenzene, diethylnitrosamine and urethane clearly show that the NORR strains are similar to the other high bioactivation strains previously described, but they lack their methodological disadvantages. We have tested six insecticides, which are characterised by having contradictory results in other genotoxicity tests, using both the standard and the high bioactivation (NORR) cross. The six insecticides analysed are the pyrethroid allethrin, the methylenedioxyphenolic compound piperonyl butoxide, the chlorinated hydrocarbons dieldrin and endrin, and the organophosphates dimethoate and malathion. We obtained negative results for all six compounds. Our results show the suitability of the wing spot test for the evaluation of compounds at the first level of genotoxicity testing.

Bioassay-directed fractionation and chemical identification of mutagens in bioremediated soils.

Soil from a Superfund site (Reilly Tar Site, St. Louis Park, Minnesota) contaminated with polycyclic aromatic hydrocarbons (PAHs) from creosote was treated with several bioremediation technologies including bioslurry (BS), biopile (BP), compost (CMP), and land treatment (LT). These treatment technologies are being evaluated in pilot scale laboratory systems by the U.S. Environmental Protection Agency's National Risk Management Research Laboratory in Cincinnati, Ohio. To evaluate the genotoxicity and identify the mutagens in the soil before and after the various treatments, fractionated extracts of five soils were bioassayed for mutagenic activity with a microsuspension modification of the Salmonella histidine reversion assay. Soils were extracted by sonication using dichloromethane (DCM). The five extracts were fractionated in triplicate (two for bioassay and one for chemical analysis) by reverse-phase high-performance liquid chromatography (HPLC) using hexane/DCM/methanol, and the fraction for bioassay were solvent-exchanged into dimethyl sulfoxide by nitrogen evaporation. Forty HPLC fractions for each sample were bioassayed in strain YG1041 with and without exogenous liver metabolic activation. As shown in a companion paper, the mutagenicity of two treatments (BS and BP) was significantly greater than the mutagenicity of the untreated soil. Mutagenic fractions (> 500 revertants) were analyzed by gas chromatography/mass spectrometry (GC/MS). PAH analysis of the soils indicated that all treatments were effective in reducing the total PAH concentration (48-74%). Qualitative GC/MS analysis of the mutagenic fractions from the BS and BP treatments indicated that they contained azaarenes, which are mutagens. The CMP and LT processes were the most effective and least toxic bioremediation procedures based on mutagenic potency and chemical analysis. This research demonstrated that the combination of bioassays and chemical analysis provided a more accurate determination of toxicity in these complex environmental mixtures.

Evaluation of the Developmental Toxicity of Caffeine and Caffeine Metabolites using the Frog Embryo Teratogenesis Assay— Xenopus (FETAX)

The developmental toxicities of caffeine and 13 metabolites, including theophylline, and paraxanthine and a synthetic methylxanthine analogue 3-isobutyl-methylxanthine (IBMX) were evaluated using the Frog Embryo Teratogenesis Assay— Xenopus (FETAX). Young X. laevis embryos were exposed to these compounds in each of two separate concentration–response experiments with and without an exogenous metabolic activation system (MAS). Results obtained from these studies indicated that relative teratogenic potencies of caffeine and each of its di- and monomethylxanthine metabolites were similar. Representatives of both the substituted uric and uracil metabolites were less developmentally toxic on an equimolar basis than the methylxanthines, suggesting that they may have represented detoxification metabolites. IBMX, a phosphodiesterase inhibitor also known to be an adenosine receptor antagonist, was the most potent developmental toxicant of the materials evaluated. In conclusion, none of the caffeine metabolites tested was found to be significantly more potent than caffeine itself in the FETAX assay.

Chemical degradation of wastes of antineoplastic agents amsacrine, azathioprine, asparaginase and thiotepa

As a part of a program devoted to the destruction of antineoplastic agents, three chemical methods readily available in the hospital environment, viz. oxidation with sodium hypochlorite (NaClO, 5%), hydrogen peroxide (H2O2, 30%), and Fenton reagent (FeCl2 · 2H2O; 0.3 g in 10 ml H2O2, 30%), were tested for the degradation of four anticancer drugs: Amsacrine, Azathioprine, Asparaginase and Thiotepa. The efficiency of the degradation was monitored by high-performance liquid chromatography. The mutagenicity of the degradation residues were tested by Ames test using tester strains Salmonella typhimurium TA 97a, TA 98, TA 100 and TA 102 with and without an exogenous metabolic activation system. Using sodium hypochlorite, 98.5% of Amsacrine, 99.0% of Azathioprine, 99.5% of Asparaginase and 98.7% of Thiotepa were destroyed after 1 hr. The hydrogen peroxide treatment destroyed 99% of Asparaginase and 98.7% of Thiotepa in 1 hr. However, this procedure was not efficient for the treatment of Amsacrine (28% after 16 hr) and of Azathioprine (53% degradation in 4 hr). The action of Fenton reagent resulted in the destruction of 98% of Amsacrine, and 99.5% of Azathioprine, 98.5% of Asparaginase and 98.7% of Thiotepa in 1 hr. In all cases where a high degree of degradation was achieved, the residues obtained were non mutagenic.

Evaluation of the Developmental Toxicities of Coumarin, 4-Hydroxycoumarin, and 7-Hydroxycoumarin Using Fetax

ABSTRACTThe developmental toxicities of coumarin and hydroxycoumarin metabolites were evaluated using FETAX. Young X. laevis embryos were exposed to coumarin, 4-hydroxycoumarin, and 7-hydroxycoumarin in each of two separate concentration-response experiments with and without an exogenous metabolic activation system (MAS) and/or inhibited MAS. The MAS was treated with carbon monoxide (CO), cimetidine (CIM), or ellipticine (ELL) to selectively modulate cytochrome P-450 activity. The MAS was also treated with cyclohexene oxide (CHO) to selectively modulate epoxide hydrolase activity. Without the MAS or inhibited MAS, coumarin and 7-hydroxycoumarin were nearly equitoxic, whereas 4-hydroxycoumarin was nearly 2-fold less developmental toxic than coumarin on an equimolar basis. Addition of the MAS and CIM-MAS increased the developmental toxicities of coumarin and, particularly, 4-hydroxycoumarin. Addition of the CHO-MAS greatly increased the developmental toxicity of coumarin and, especially, 4-hydroxycoumarin. Addition of the ELL- or CO-inhibited MAS did not increase the developmental toxicity of coumarin. However, addition of the intact MAS did not alter the developmental toxicity of 7-hydroxycoumarin. Results from these studies suggested that P-450; specifically ELL-inhibited P-450 (arylhydrocarbon hydroxylase) may have been responsible for increasing the developmental toxicity of coumarin. Furthermore, the increased toxicity of coumarin or 4-hydroxycoumarin following co-incubation with CHO-treated microsomes indicated that highly toxic epoxide intermediates may be produced from oxidative P-450 metabolism and that epoxide hydrolase may play a role in detoxification of the reactive intermediates.

Phase III Interlaboratory Study of Fetax, Part 2: Interlaboratory Validation of an Exogenous Metabolic Activation System for Frog Embryo Teratogenesis Assay-Xenopus (Fetax)

ABSTRACTInterlaboratory validation of an exogenous metabolic activation system (MAS) developed for the alternative, short-term developmental toxicity bioassay, Frog Embryo Teratogenesis Assay - Xenopus (FETAX) was performed with cyclophosphamide and caffeine. Seven study groups within six separate laboratories participated in the study in which three definitive concentration-response experiments were performed with and without the MAS in a side-by-side format for each chemical. Since both chemicals had been previously tested in FETAX, the test concentrations were provided to each laboratory prior to testing. Interlaboratory coefficient of variation (CV) values for unactivated cyclophosphamide (no MAS) were 15%, 15%, 29%, and 25% for the 96-hr LC50, 96-hr EC50 (malformation). Minimum Concentration to Inhibit Growth (MCIG), and Teratogenic Index (TI) values, respectively. Addition of the MAS increased the CV values of each endpoint at least 3.9-fold. Interlaboratory CV values for unactivated caffeine were 31%, 18%, 31%, and 46% for the 96-hr LC50, 96-hr EC50 (malformation), MCIG, and TI values, respectively. Addition of the MAS decreased the CV values of each respective endpoint by at least 1.6-fold. Results indicated that bioactivated toxicants may be prone to greater variability in response amongst laboratories than compounds, which are detoxified. Even though more variability was noted with activated cyclophosphamide, results were within interlaboratory variation expected for other aquatic-based bioassays. Thus, results from these studies warrant the continued use and further refinement of FETAX for alternative developmental toxicity assessment.

In vitro mutagenicity and genotoxicity study of a number of short-chain chlorinated hydrocarbons using the micronudeus test and the alkaline single cell gel electrophoresis technique (Comet assay) in human lymphocytes: a structure–activity relationship (QSAR) analysis of the genotoxic and cytotoxic potential

Using the micronucleus (MN) test and the alkaline single cell gel electrophoresis (Comet) assay, potential mutagenicity (MN formation), genotoxicity (DNA breakage capacity) and cytotoxicity (cell proliferation reduction) of five chlorinated hydrocarbons (carbon tetrachloride, hexachloroethane, 1,2-dichloroethane, 1-chlorohexane and 2,3-dichlorobutane) have been evaluated in isolated human lymphocytes. With the MN test a low but statistically significant mutagenic activity was detected for all tested substances (except 2,3-dichlorobutane) with one out of the two donors and in the presence or absence of an exogenous metabolic activation system (S9 mix). However, at the concentration ranges tested none of the positive compounds induced a clear dose-dependent mutagenic effect. The Comet assay detected a strong DNA damaging effect for 1-chlorohexane, 2,3-dichlorobutane and 1,2-dichloroethane, but not for carbon tetrachloride and hexachloroethane. The influence of metabolism on the genotoxic activity of the chemicals was more clear in the Comet assay than in the MN test. The experimental genotoxicity and cytotoxicity data obtained in this study, together with data on five more related chemicals previously investigated, and their physico-chemical descriptors or electronic parameters have been used for QSAR analysis. The QSAR analysis high-lighted that the toxicity of the tested compounds was influenced by different parameters, like lipophilicity (logP), electron donor ability (charge) and longest carbon-chlorine (LBC-Cl) bond length. In addition, steric parameters, like molar refractivity (MR) and LBC-Cl, and electronic parameters, like ELUMO (energy of the lowest unoccupied molecular orbital, indicating electrophilicity), were predominant factors discriminating genotoxins from non-genotoxins in the presence but not in the absence of S9 mix. Although a limited number of compounds have been examined and cytotoxicity and genotoxicity were identified in two different bioassay tests, the data set was obtained by the same experimentor, strengthening the reliability of the QSAR.