Analysis of the mechanism of isoniazid-induced developmental toxicity with frog embryo teratogenesis assay: Xenopus (FETAX)

The developmental toxicity of isoniazid (INH) and the metabolites acetylhydrazide (AH) and isonicotinic acid (INA) were examined with the frog embryo teratogenesis assay-Xenopus (FETAX). Late Xenopus laevis blastulae were exposed to INH, AH, and INA for 96 h in two separate static-renewal tests with and without the presence of three differently induced metabolic activation systems (MAS). The MAS consisted of uninduced, Aroclor 1254-induced, and INH-induced rat liver microsomes. Addition of the INH-induced MAS decreased the 96 h LC50 of INH and AH approximately 1.6-fold and 7.9-fold, respectively. The 96 h EC50 (malformation) of INH was virtually unaffected; however, the INH-MAS decreased the teratogenic index (TI) [96 h LC50/96 h EC50 (malformation)] nearly 1.8-fold. The 96 h EC50 (malformation) of AH increased approximately 2.0-fold, decreasing the teratogenic index value 15.8-fold. INA yielded a teratogenic index value of 2.5. Neither the uninduced MAS nor the Aroclor 1254-induced MAS had an effect on any of the compounds tested and none of the MAS affected the developmental toxicity of INA. Results from this study suggest that mixed functional oxidase metabolism may alter the developmental toxicity of INH in vitro by producing a more embryolethal, but less teratogenic metabolite(s) than INH or AH themselves. Results are indicative of the utility and versatility of FETAX in evaluating toxicological mechanisms of teratogenesis in vitro.     

Evaluation of the developmental toxicity of nicotine and cotinine with frog embryo teratogenesis assay: Xenopus

The teratogenic potential of nicotine and a primary metabolite, cotinine, was examined with FETAX (Frog Embryo Teratogenesis Assay: Xenopus). Early embryos of Xenopus laevis were exposed for 96 hr to nicotine or cotinine in two separate static renewal tests of each compound without addition of the metabolic activation system (MAS). Two static renewal tests of nicotine with the MAS were also conducted. Addition of the MAS to nicotine reduced the LC50 from an average of 136 to 20 mg/L. However, the EC50 (malformation) was increased from 0.4 to 5.8 mg/L upon activation. The LC50 and EC50 values for cotinine averaged 4,340 and 720 mg/L, respectively. Based on mortality/malformation index values, growth end points, and the types and severity of the induced malformations, nicotine and cotinine scored as potential teratogens. Metabolism of nicotine to more polar metabolites increased the nicotine concentration required to induce terata. The results are indicative of the versatility of FETAX in developmental toxicity testing.     

Metabolic activation in the fetal mouse salivary gland culture system with rat hepatocytes, rat S-9, and human S-9.

The usefulness of an in vitro assay for embryotoxicity may depend on the availability of metabolic activation systems that will function in the culture system. The fetal mouse salivary gland has been investigated as an in vitro assay system. To see if the glands would grow in the presence of metabolic activators and if the glands would react to metabolites known to be embryotoxic, the glands were grown in the presence of cyclophosphamide (CP) and several activation systems. These included isolated rat hepatocytes, uninduced rat S-9, rat S-9 induced with 3-methylcholanthrene (3-MC), rat S-9 induced with Aroclor 1254, and human S-9. Twenty salivary glands were isolated from 13 day embryos (plug day = 0) and were grown in each treatment for 48 h. One control had no activation system of CP, one had an activation system but no CP, and three treatments had the activation system and 25, 75, or 150 micrograms/ml CP. The S-9 with cofactors and the appropriate amount of CP was contained in dialysis bags. The greatest suppression of salivary gland growth occurred in co-culture with hepatocytes activating CP. The S-9 induced by Aroclor 1254 was nearly as effective as the hepatocytes. The next most effective was a group with similar activity consisting of the uninduced rat S-9 and the three samples of human S-9. The 3-MC-induced S-9 was the least effective in suppressing growth of salivary glands. All the activation systems tested can be used with the salivary gland culture system.     

Analysis of the activity of DNA, RNA, and protein synthesis inhibitors on Xenopus embryo development

The teratogenic and growth-inhibiting potential of DNA, RNA, and protein synthesis inhibitors was explored using the Frog Embryo Teratogenesis Assay: Xenopus (FETAX). Endpoints measured in 96-h static tests were survival, malformation, ability to swim, skin pigmentation, stage of development, and growth. The DNA synthesis inhibitors hydroxyurea, cytosine arabinoside, and ethidium bromide proved to be teratogenic by the severity of malformations induced. Hydroxyurea gave an LC50 of 1.82 mg/ml, an EC50 (malformation) of 0.43 mg/ml, while the values for cytosine arabinsode were 5.41 and 0.76, respectively. The values for ethidium bromide were 0.05 and 0.035. The RNA synthesis inhibitor actinomycin D and the protein synthesis inhibitor cycloheximide were more embryolethal than teratogenic but significantly inhibited growth as determined by head-tail length measurements. Actinomycin D caused severe malformations, while cycloheximide caused relatively minor abnormalities. The LC50 for actinomycin D was 1.89 mg/ml, while the EC50 (malformation) was 2.17 mg/ml. For cycloheximide, the values were 1.59 and 1.19, respectively. FETAX advantages include rapid data collection, the ability to measure stage-dependent effects, and the ability to use a large number of embryos to obtain excellent dose-response curves with narrow confidence limits. Disadvantages include lack of a metabolic activation system, absence of a placental relationship, and the inability to detect specific abnormalities such as limb defects in 96 h.     

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.     

Assessing the predictive validity of frog embryo teratogenesis assay-Xenopus (FETAX)

The ability of frog embryo teratogenesis assay - Xenopus (FETAX) to identify the potential developmental toxicity of a group of diverse chemicals was evaluated by comparison with results from in vivo studies in rats. A total of 12 chemicals, three of which were shown to be teratogenic in vivo, four of which were embryolethal (but not teratogenic) in vivo, and five which did not produce any developmental toxicity in vivo in the rat were evaluated using FETAX. Results of the FETAX test with these 12 blind-coded compounds correctly predicted that three chemicals had strong teratogenic potential, four had low teratogenic hazard potential but were embryolethal, and five posed little if any developmental toxicity hazard. In addition, this study concluded that within a family of chemistry analogs could be ranked according to relative teratogenic hazard and that for the teratogenic compounds the types of malformations induced in Xenopus mimicked the abnormalities induced in vivo in rats. In summary, these results confirmed that the FETAX assay is predictive and can be useful in an integrated biological hazard assessment for the preliminary screening of chemicals. Teratogenesis Carcinog. Mutagen. 20:87-98, 2000.     

Bioactivation of 6-aminochrysene by animal and human hepatic preparations: contributions of microsomal and cytosolic enzyme systems

6-Aminochrysene was converted into mutagen(s), in the Ames testin the presence of Aroclor 1254-induced hepatic S9, microsomaland cytosolic fractions, the first being the least and the lastthe most efficient activation system. The cytosolic activationof 6-aminochrysene decreased in the presence of increasing amountsof microsomes. The Aroclor 1254-induced rat microsomal and cytosolicsystems differed markedly in a number of properties, includingtheir cofactor requirements and responses to prototype inducersof the cytochrome P450-dependent mixed-function oxidase system.The cytosolic activation system could also convert 2-aminochryseneto mutagens but not 2- and 6-methylchrysene. Human hepatic cytosolcould convert 6-aminochrysene and 2-aminoanthracene to mutagensin the Ames test. It is concluded that a hepatic cytosolic oxygenaseexists, totally different from the microsomal oxygenases, whichmetabolizes aminopolycyclic aromatic hydrocarbons to mutagens,presumably through N-oxidation. This oxygenase activity appearsto be present in human hepatic cytosol. ¹To whom correspondence should be addressed     

The structure--activity relationships of flavonoids as inhibitors of cytochrome P-450 enzymes in rat liver microsomes and the mutagenicity of 2-amino-3-methyl-imidazo[4,5-f]quinoline

The antimutagenicity of 19 naturally occurring flavonoids andtheri derivatives including flavones, flavonols, flavanoes,inoflavones and flavanols were determined using Salmonella typhimuriumTA98 against 2-amino-3-methylimidazo[4,5-f] quinoline (IQ) inthe presence of Aroclor 1254-induced rat hepatic S9. In general,a relationship between the chemical structure of flavonoidsand their antimutagenicity was found for compounds containingone or more of the following featutes: (i) C4 keto group, (ii)aglycone, (iii double bond at positions C2 and C3, (iv) phenylgroup at position C2, and (v) three hydroxyl substituents atpositons C4', C5 and C7. The inhibitory effects of flavonoidson activities of 7-ethoxycoumarin deethylase (ECD) and 7-ethoxyresorufindeethylase (ESD) of Aroclor 12540-induced hepatic microsomeswere also examined. In addition, we studied the effects of flavonoidson the metabolism of IQ by Aroclor 1254-induced microsomes usinghigh-performance liquid chromatography. The antimutagenicitycorrelated with the inhibition of cytochrome P-450IA1-linkedESD and P-450IA2-linked ECD activity in hepatic microsomes,and with an inhibition of N-hydroxy-IQ fromation from IQ metabolismby hepatic microsomes. These reslute indicated that flavonesor flavonols that contasin C5, C7 and C4' hydroxyl groups arepotent inhibitors of P-450 enzyme activities induced by Aroclor1254 (P-450IA1 and P-450IA2), and masy potentially be usefulas chemopreventive agents against heterocyclic amine-inducedmutagenesis or carcinogenesis. ¹To whom correspondence sould be addressed     

Morphological transformation of BALB/3T3 cells by various procarcinogens in the presence of a rat liver S-9 activation system

An Aroclor-induced rat hepatic S-9 metabolic activation system was incorporated into the BALB/3T3 cell transformation assay to increase its sensitivity to a wide range of procarcinogens. S-9 was prepared from Aroclor 1254-induced (500 mg/kg) Fischer 344 rats. Cyclophosphamide, dimethylnitrosamine, 2-aminofluorene, and 2-naphthylamine were metabolized to reactive forms capable of inducing both dose-dependent toxicity and morphological transformation of BALB/3T3 cells. Treatments without an exogenous metabolic activation system were nontoxic and nontransforming. Adaptation of this commonly used exogenous metabolic activation system to BALB/3T3 cells will allow detection of the transforming potential of procarcinogens which test negative in a standard assay.     

Antimutagenicity of Maillard reaction products from amino acid/sugar model systems against 2-amino-3-methylimidazo-[4,5-f]quinoline: the role of pyrazines

The antimutagenicity of dichloromethane extracts from eightamino acid/sugar model systems was determined using Salmonellatyphimurium TA98 against 2-amino-3-methyl-imidazo[4,5-f]quinoline(IQ) in the presence of Aroclor 1254-induced rat hepatic S9.The Maillard reaction products in the dichloromethane extractswere then quantified and qualified by capillary gas chromatographyand gas chromato-graphy-mass spectrometry, respectively. Pyrazinesand furans were found to be the major Maillard reaction productsyielded in the extracts. Moreover, the antimutagenicity of dichloromethaneextracts correlated positively with the total amounts of pyrazinesand furans. To elucidate the mechanism of antimutagenicity ofdichloromethane extracts, the inhibitory effect of pyrazineson ethoxycoumarin deethylase activity in Aroclor 1254-inducedhepatic microsomes was examined. We also studied the effectsof pyrazines on IQ metabolism by Aroclor 1254-induced microsomesusing high-performance liquid chromatography. The antimutagenicityof pyrazines correlated positively with both the inhibitionof cytochrome P-450 IA2-linked ethoxycoumarin deethylase inhepatic microsomes and the inhibition of N-hydroxy-IQ formationfrom IQ metabolism by hepatic microsomes. Thus we concludedthat pyrazines in dichloromethane extracts from eight aminoadd/sugar model systems play an important role in the antimutagenicityof IQ. Moreover, we concluded that the modifying effect of pyrazineson the mutagenicity of IQ is mediated through interaction withmicrosomal activating enzymes to inhibit the major active metabolitein N-hydroxy-IQ formation. ¹To whom correspondence should be addressed     

Co-culture of rat embryos and hepatocytes: in vitro detection of a proteratogen

The technique of whole embryo culture developed by New [Environ Health Perspect 18:105-110, 1976] provides a sensitive assay to evaluate the effects of a test chemical on embryo development independent of maternal influences. To detect proteratogens, this assay must be coupled with an exogenous metabolic activation system. We have developed methods for the co-cultivation of rat embryos with primary hepatocytes, which offers several advantages over subcellular fractions when providing metabolic activation for in vitro assays. In the present study, rat embryos removed from the dam on day 10 of pregnancy were co-cultivated in vitro with primary cultures of rat, rabbit, or hamster hepatocytes. Embryos co-cultivated with hepatocytes developed normally, as did embryos exposed to a test chemical, cyclophosphamide (CP) in the absence of hepatocytes. When embryos were co-cultivated with hepatocytes and exposed to CP, a dose-related embryotoxicity was observed, indicating metabolic activation of the proteratogen. Using hepatocytes isolated from rats pretreated in vivo with phenobarbital, we observed an increase in CP-induced malformations and embryotoxicity compared to those of embryos exposed to CP in the presence of uninduced hepatocytes. The teratogenic bioactivation of CP was inhibited in vitro by the addition of metyrapone. When similar numbers of hepatocytes were used for metabolic activation of CP the induced embryotoxicity was greater in the presence of rabbit and hamster hepatocytes than with rat hepatocytes. Development of procedures for the culture of rat embryos with hepatocytes from other species suggests the utility of this in vitro system for the investigation of species differences in sensitivity to chemical teratogens.     

Cytosolic potentiation of the rat hepatic microsome mediated mutagenicity of benzidine

The role of the cytosolic fraction in the S9-mediated metabolicactivation of benzidine to mutagens in the Ames test was investigatedusing hamster and rat hepatic preparations. Rat microsomes alonewere poor activators of benzidine compared to hamster microsomes,at least partly explaining the well known superiority of S9preparations from the latter species in activating this amine.Supplementation of rat microsomal preparations with the cytosolicfraction from hamsters and to a lesser extent from rats enhancedthe bioactivation of benzidine. When hamster microsomal preparationswere supplemented with rat or hamster cytosolic fractions nosignificant effect was observed. Cytosolic fractions from eitherspecies could not activate benzidine to mutagens in the absenceof microsomes. The cytosolic potentiation of the microsome-mediatedactivation of the amine was not inducible by Aroclor 1254. Similarly,the microsome-mediated activation of benzidine was not enhancedby Aroclor 1254 pre-treatment of the rats, when mutagenicityis expressed per nmol of cytochrome P-450. It is concluded that(i) the cytosolic fraction may play an important role in themetabolic activation by S9 preparations of chemical carcinogenssuch as benzidine, and (ii) the Aroclor-induced isozymes ofcytochrome P-450 do not catalyse the N-hydroxylation of benzidine.     

Teratogenicity of cobalt chloride in Xenopus laevis, assayed by the FETAX procedure.

The teratogenicity of cobalt chloride (CoCl2) was tested by the FETAX (Frog Embryo Teratogenesis Assay: Xenopus) procedure in the South African frog, Xenopus laevis. In five assays, beginning at 5 h post-fertilization, groups of Xenopus embryos were incubated for 96 h in media that contained CoCl2 at concentrations ranging from 1.8 x 10(-6) to 1.8 x 10(-2) mol/L; control groups were incubated in the same medium without added CoCl2. At 101 h post-fertilization, surviving embryos were counted, fixed in formalin, and examined by microscopy to score malformations and measure head-to-tail lengths. In control embryos, survival was greater than or equal to 95% and malformations were less than or equal to 5%. Malformations were found in greater than 99% of embryos exposed to Co2+ levels greater than or equal to 56 mumol/L. Co2+)-exposed embryos showed a concentration-related pattern of malformations, comprising gut malrotation, ocular anomalies, kinked tail, craniofacial dysplasia, cardiac deformities, and dermal blisters. Other concentration-dependent abnormalities, not categorized as malformations, included stunted growth, edema, ventral distention, and hypopigmentation. The median embryolethal concentration (LC50) of CoCl2 was 10.4 (SE +/- 0.4) mmol/L; the median teratogenic concentration (EC50) was 25 (SE +/- 2) mumol/L; the teratogenic index (TI = LC50/EC50) was 416 (SE +/- 13), indicating that CoCl2 is a potent teratogen for Xenopus laevis.     

Effects of black tea theafulvins on aflatoxin B(1) mutagenesis in the Ames test

Black tea theafulvins, a fraction of thearubigins isolated from black tea aqueous infusions, potentiated the mutagenic activity of the mycotoxin aflatoxin B(1) in the Ames test, in the presence of a hepatic S9 activation system derived from Aroclor 1254-treated rats. In contrast, when the S9 activation system was replaced with isolated microsomes, theafulvins suppressed the mutagenicity of the mycotoxin. When microsomal metabolism was terminated after metabolic activation of the mycotoxin, incorporation of the theafulvins into the activation system reduced the mutagenic activity, whereas if it was added before termination of microsomal activity a potentiation of mutagenic response was observed. In in vitro studies, theafulvins inhibited epoxide hydrolase and glutathione S-transferase activities in a concentration-dependent manner. Finally, the mutagenicity of aflatoxin B(1) was much more pronounced in bacteria that were pre-exposed to theafulvins but from which they were subsequently washed off. It may be inferred from the above studies that the genotoxic synergy between aflatoxin B(1) and black tea theafulvins does not occur during the bioactivation of the carcinogen, but may partly be due to decreased deactivation of the reactive intermediate, aflatoxin B(1) 8,9-oxide, by conjugation with glutathione.     

Metabolic activation of 3-(2-chloroethoxy)-1,2-dichloropropene: a mutagen structurally related to diallate, triallate, and sulfallate

3-(2-Chloroethoxy)-1,2-dichloropropene (CP), a Salmonella promutagen that was recently isolated from a sample of residue organics previously concentrated from drinking water, is structurally related to three other chlorinated promutagens, the S-chloroallyl thiocarbamate herbicides diallate, triallate, and sulfallate. These four chloroallyl ether compounds were found to be similar with respect to strain specificity, potency, and requirement for specific metabolic activation. The 9,000g supernatant (S9) fractions from polychlorinated biphenyl Aroclor 1254- or phenobarbital-induced rats metabolized the four chloroallyl ethers to mutagenic products, whereas S9 from 3-methylcholanthrene-induced or uninduced rats did not. The metabolic activation of CP, diallate, and triallate to mutagens was catalyzed by the 100,000g microsomal pellet of S9 alone, but the activation of sulfallate to mutagenic metabolites required both microsomal and cytosolic fractions of S9. Direct-acting (minus S9) mutagenic metabolites of diallate and triallate could be extracted into methylene chloride from S9 incubation mixtures. Incubations containing S9 and either sulfallate or CP did not yield methylene chloride-extractable metabolites with direct-acting mutagenic activity. On the basis of these results and those from previous studies on the metabolism of diallate, triallate, and sulfallate, a tentative model for the metabolic activation of CP is proposed in which this chloroallyl ether undergoes alpha-carbon hydroxylation to form multiple mutagenic products.     

In vitro embryotoxic effects of ethylene glycol in rats

Rat embryos of the CD strain were treated in a whole embryo culture system with either 30 or 40 microliters of ethylene glycol (EG) per milliliter of culture medium for the first 8 (0-8 hr) or second 8 (8-16 hr) hr of a 48-hr culture period. The compound was not embryolethal under these conditions but did alter growth and development. EG at 40 microliters/ml of culture medium at 8-16 hr decreased morphological score, somite number, crown-rump and head lengths, as well as DNA and protein contents. The most frequent abnormality induced by the compound was absence of yolk sac circulation; absent hindlimb bud, hypoplastic telencephalon, and lack of development of the otic and optic systems were also seen in EG-exposed embryos. Since it has been reported that rodent embryos cultured in vitro lack alcohol and aldehyde dehydrogenases that metabolize EG, present results suggest that the parent compound is capable of altering normal embryonic development when administered during a brief period or organogenesis.     

Cytotoxicity and mutagenicity of dimethylnitrosamine in mammalian cells (CHO/HGPRT system): Enhancement by calcium phosphate

The cytotoxicity and mutagenicity of dimethylnitrosamine (DMN) was determined in the CHO/HGPRT system. Metabolic activation of the promutagen was achieved by use of a liver homogenate supernatant (S9) prepared from Aroclor 1254-induced Sprague-Dawley rats. The cytotoxic and mutagenic effects of DMN were enhanced by the inclusion of calcium chloride in the incubation mix, and this enhancement was dependent on the presence of sodium phosphate. Under conditions that yielded maximal activity (10 mM calcium chloride, 10 mM magnesium chloride, 50 mM sodium phosphate), an apparent calcium phospate precipitate was observed. DMN activity increased with increasing amounts of S9 protein over the range 0.3–3.0 mg/ml in the S9 mix and appeared to plateau at higher concentrations. The mutagenicity of DMN can be described as 110 mutants/10⁶ cells per mM DMN per mg/ml S9 protein per hour.     

Alternatives to Aroclor 1254-induced S9 in in vitro genotoxicity assays.

A working party was set up by the UK Environmental Mutagen Society to consider alternatives to Aroclor 1254 (Aroclor)-induced S9 in in vitro genotoxicity assays, with the aims of considering whether a replacement for Aroclor in its role in general screening assays could be readily identified. The working party concluded that there was sufficient support in the literature to justify the use of an appropriate phenobarbital/beta-naphthoflavone regime as an acceptable alternative to Aroclor.     

Cyclophosphamide teratogenesis: a review

Cyclophosphamide (CP) is one of the best studied teratogens; it produces primarily central nervous system and skeletal anomalies in rats, mice, rabbits, monkeys, and humans. Furthermore, CP is one of the most extensively studied antineoplastic agents. Recent work using in vitro rodent embryo culture has demonstrated that CP must be bioactivated to be teratogenic. This finding extends earlier work showing that CP must be activated to achieve its antineoplastic and mutagenic effects. Activation of CP to its teratogenic, mutagenic, and antineoplastic form is mediated by microsomal cytochrome P-450 monooxygenases, which convert CP to 4-hydroxycyclophosphamide (4OHCP). In the absence of detoxification, 4OHCP spontaneously breaks down to phosphoramide mustard (PM) and acrolein (AC). PM is the CP metabolite believed to be responsible for the antineoplastic and mutagenic effects of CP, whereas AC is thought to cause the side effects associated with CP chemotherapy. Recent work has shown that the teratogenic effects of CP are mediated by both PM and AC. Although it is far from proven, available evidence supports the hypothesis that DNA is the primary target in terms of the teratogenic, mutagenic, and antineoplastic effects of CP. Although the nature of the DNA lesions produced by CP, which are responsible for its teratogenic, mutagenic, and antineoplastic effects, is not completely understood, cross-linking of DNA seems to play a critical role in the antineoplastic properties of CP. Preliminary information obtained from embryos exposed to CP metabolites suggests that, although DNA cross-linking might play a role in CP teratogenesis, metabolite-induced DNA strand breakage and/or induction of mutations might also play a role. Although insights into the molecular mechanisms underlying CP teratogenesis are just beginning to accumulate, the availability of in vitro embryo culture combined with the modern armamentarium of molecular biology will allow teratologists to probe further the molecular aspects of teratogenesis.