Environmental Health PerspectivesVol. 70, pp. 131-136, 1986

The Potential Role of Redox Cycling as aMechanism for Chemical Teratogenesisby Mont R. Juchau,* Alan G. Fantel,t Craig Harris,* andBruce K. Beyer*

A survey of the literature indicates that several chemicals whose reduced metabolites are capable ofundergoing redox cycling in biological systems also possess significant teratogenic properties when testedin vivo. We have initiated investigations to determine whether the embryotoxic effects of such chemicalscould result from their redox cycling properties and whether redox cycling could be an important mech-anism in chemical teratogenesis. In order to obviate the potentially confounding influences of maternalfactors, our initial studies have been performed with a whole embryo culture system with redox cyclingagents added directly to the culture medium. Several representative redox cycling agents including dox-orubicin, paraquat, a series of nitroheterocycles, nitrosofluorene, and diethylstilbestrol (converted met-abolically to redox cycling quinone/semiquinone radicals) have been investigated thus far. The nitrohet-erocycles which bear nitro groups with comparatively high redox potentials produced a striking,asymmetric defect involving primarily the right half of the prosencephalic and mesencephalic regions.The effect was exacerbated under conditions of low 02 tension. Accumulated data to date strongly suggestthat reduction of the nitro group is an essential feature in the embryotoxic mechanism. Quinones (dox-orubicin, paraquat) and compounds metabolically converted to quinones (diethylstilbestrol) appeared toproduce embryotoxic effects via mechanisms not associated with redox cycling. Nitrosofluorene embry-otoxicity was markedly exacerbated by changes in both intra- and extracellular glutathione levels, butdefinitive dependence on a radical-mediated effect or redox cycling was not demonstrated.

IntroductionThe biochemistry of chemical teratogenesis is a rel-

atively new area of research that remains largely unex-plored (1). Because of the intricate nature ofmorphogenesis, the complexity of the problems asso-ciated with such research is enormous and undoubtedlydiscourages many potential investigators who otherwisecould contribute to a better understanding of mechan-istic aspects. Thus much ofthe existing information con-cerning chemically elicited developmental abnormalitiesis still of a more descriptive than mechanistic nature.Some recent, fundamental scientific advances, however,should encourage researchers to explore more seriouslythe biochemical mechanisms whereby chemicals canelicit developmental structural anomalies. The evolutionof a successful whole embryo culturing system repre-sents a highly significant technological advance and nowpermits investigators to study the effects of chemicalson mammalian prenatal development in the absence ofpotentially confounding maternal factors. The culturesystem has also enabled a full recognition of the impor-tance of both maternal and embryonic biotransforma-

*Department of Pharmacology, School of Medicine, University ofWashington, Seattle, Washington 98195.tDepartment of Pediatrics, School of Medicine, University ofWash-

ington, Seattle, WA 98195.

tion/bioactivation as critical determinants of the capac-ity of chemicals to elicit defects (2). Ascertainment ofthe identities of ultimate chemical dysmorphogenicagents and of regulatory factors governing their gen-eration during organogenesis will undoubtedly assist inelucidating teratogenic mechanisms. Thus far, focus inthis research area has been on the cytochrome P-450-dependent generation of reactive intermediates by bothextraembryonic (3-6) and embryonic (7-9) enzymesources. The purpose of this article is to discuss someinitial and preliminary findings with respect to cyto-chrome P-450-independent embryonic systems that ap-pear capable ofconverting potential prodysmorphogenicagents to their ultimate, biologically active forms. Mostof the drugs and chemicals in this category that havebeen investigated thus far are those that can undergoreduction by cellular reducing agents followed by reox-idation by molecular oxygen (02). Reoxidation resultsin the generation of the original, parent compound plussuperoxide anion (02) and, subsequently, highly reac-tive metabolic derivatives of oxygen. Such chemicalsare also known as "redox cyclers" and may producebiological effects via a variety of consequences of theredox cycling process (10). Agents to be discussed willinclude doxorubicin (Adriamycin), a series of hetero-cyclic nitro compounds, certain nitroarenes, paraquat,menadione, diethylstilbestrol (DES), nitrosofluorene,

JUCHAU ET AL.

and 2-acetylaminofluorene. Each ofthese substances (ortheir respective metabolites) is known to be capable ofundergoing redox cycling in biological systems and hasalso been studied in terms of its embryotoxicity in thewhole embryo culture system in our laboratories. Itshould be re-emphasized that studies performed as ofthis writing have only very recently been initiated andmany are thus of a preliminary nature. They representthe first reported attempts to investigate redox cyclingas a potential mechanism of chemically induced terata.Therefore, any conclusions drawn therefrom must beregarded at present as strictly tentative and/or hypo-thetical.

Redox Cycling: The PhenomenonRedox cycling agents are chemicals that are capable

of undergoing single electron reduction reactions toyield radical species (10). Such agents, in accordancewith their respective redox potentials, can accept elec-trons from a variety of biological reducing agents in-cluding reduced flavoproteins, reduced ferredoxin,NADPH, NADH, reduced glutathione (GSH) and otherthiol-containing compounds, ascorbate, and probablyothers. Enzymes known to catalyze such reactions arefrequently flavoproteins and include NADPH-cyto-chrome P-450 reductase, xanthine oxidase, mitochon-drial flavoproteins of the electron transport chain andvarious other dehydrogenases. Frequently, redox cy-cling xenobiotics accept electrons directly from reducedflavoproteins.

In the presence of 02, the reduced organic chemicalcan be reoxidized to the original parent compound bydonating a single electron to 02 thus converting it to02. As long as both 02 and suitable reducing equiva-lents (e.g., NADH, NADPH, GSH) are available, therepetitive reduction and reoxidation reactions result inthe occurrence of a redox cycle and the process is re-ferred to as "futile cycling." Clearly, this process (Fig.1) could conceivably elicit cytotoxicity via depletion ofessential reducing equivalents, depletion of 02 (partic-ularly if involved cells are already living under some-

what hypoxic conditions) or via interactions of reactiveorganic radical forms with critical biological macromo-lecules. Interaction with small biomolecules is also apotential damaging factor. GSH, for example, can com-pete with 02 for nitro anion radicals (11), and redoxcycling can thus lead to depletion of the reduced formof cellular glutathione which constitutes one of the cell'smost important defenses against cytotoxic agents andalso subserves several highly important biological func-tions (DNA synthesis, etc.). Such organic radicals canalso initiate lipid peroxidation reactions resulting in thedisruption or disorganization of cellular membranes andultimate cell death if sufficiently extensive.In addition, generation of 02 leads to the formation

of hydrogen peroxide, both spontaneously and via thecatalytic action of superoxide dismutase. In the pres-ence of transition metals (Fe2+, Cu+) and under otherappropriate conditions, hydrogen peroxide, in turn, canbe converted to highly reactive and potentially verytoxic hydroxyl radicals (- OH) and to singlet oxygen.Under conditions in which 02 tensions are relativelyhigh, toxicity resulting from the generation of hydroxylradicals and/or from the depletion ofimportant reducingequivalents may predominate whereas, under condi-tions of low 02 tension, the major cause of toxicity maybe an exacerbation of the hypoxic state and/or an in-crease in the steady-state levels of organic radicals.Radical species may bind covalently to critical macro-molecules, initiate free radical-mediated reactions suchas lipid peroxidation or act via less well-establishedmechanisms.To what extent do redox cycling chemicals exist in

the modern environment? Prototype redox cyclingagents currently undergoing intensive investigation arearomatic nitro compounds (12) and quinone/quinonoids(13). The aromatic nitro group can undergo single-elec-tron reduction to yield nitro anion radicals that readilyreduce 02 to 02. Their capacity to undergo redox cy-cling under conditions of relative hypoxia has been im-plicated in their antimicrobial, radiosensitizing, carcin-ogenic, and mutagenic properties. Nitro compounds canbe further reduced to the corresponding nitroso analogs

Breoks

FIGURE 1. Metabolic pathways involved in the redox cycling of foreign organic chemicals (10).

132

REDOX CYCLERS AS TERATOGENS

that may undergo redox cycling via conversion to ni-troso anion radicals. Other radical species feasibly maybe generated during the ultimate reduction to primaryamines via the hydroxylamine, itself a reactive, toxicintermediate.

Quinones may undergo either one- or two-electronreduction reactions. The former, resulting in the for-mation of radical semiquinones, represents a bioacti-vation reaction due primarily to the reactivity of thehighly unstable semiquinone radical. The two-electronreduction reactions, catalyzed by diaphorases, result inthe formation of fully reduced hydroquinones which ap-pear to be virtually inactive biologically and are nor-mally regarded as detoxication processes. One of themost notorious teratogenic chemicals, DES, appears ca-pable of producing various deleterious biological effectsby virtue of its conversion to quinones and semiquinones(14-16).Other foreign organic chemicals that accept single

electrons in a stepwise fashion and are thus convertedto free radical intermediates potentially capable of re-dox cycling are azo derivatives, nitroso compounds, N-oxides, S-oxides, and polyhalogenated aliphatic hydro-carbons (10). Carbonyl compounds also undergo dehy-drogenase-catalyzed reduction reactions but these aretwo-electron reduction reactions that do not involve theproduction of radical intermediates. Living cells havestrongly negative redox potentials and are thus capableof readily reducing each of these classes of chemicals.A large number of factors are important determinantsof the quantitative generation of biologically active, re-active intermediates from these chemicals. These in-clude the redox potential of the reducible organic chem-ical and the availability and activity of enzymes thatcatalyze their respective reduction reactions. Also, theenergy status and 02 tension of potentially affected cellsare clearly major factors. Under certain conditions, theavailability of reducing equivalents and cellular concen-trations of antioxidants (a-tocopherol, ascorbate, reti-noids, GSH, uric acid, etc.) can also be of considerableimportance. Clearly, the manner in which the conceptushandles such chemicals and the embryotoxicity causedby these agents should be a matter of highest impor-tance and interest.

Redox Cyclers as Teratogens inVivoWhich chemicals known or reputed to produce bio-

logical effects via redox cycling are also reasonably wellestablished teratogens or embryotoxins? Some of themore well known redox cyclers with reported terato-genic properties are listed in Table 1. Currently, thereis no evident relationship between redox cycling andthe qualitative nature of malformations produced invivo. Clearly, many of the qualitative differences ob-served thus far might be accounted for in terms of dis-positional factors. For example, a number of the com-pounds listed in Table 1 do not cross membranes readily,

whereas others are distributed rather evenly through-out biological systems. Trypan blue (actively excludedfrom most cells) is an azo dye reputed to produce ter-atogenic effects by virtue of a specific action on thevisceral yolk sac (17). It would be of interest to deter-mine whether other highly polar redox cyclers mightproduce teratogenic effects similar in nature to thoseelicited by trypan blue. Additionally, the toxic effectsof some redox cyclers (e.g., bleomycin, streptonigrin)are exacerbated (10) by high 02 tension (tending to im-plicate reactive oxygen metabolites) whereas the mostprofound effects of other redox cyclers (e.g., misoni-dazole, mitomycin c) are observed under conditions oflow 02 tension. Thus, even if it were to be presumedthat the chemicals listed in Table 1 produced their ter-atogenic effects exclusively via redox cycling, one wouldnonetheless expect these agents to elicit a variety ofdifferent abnormalities as a result of differences in dis-position and chemical properties. It must be borne inmind, of course, that the teratogenic effects producedmay result from actions entirely independent of redoxcycling.

Effects of Redox Cyclers onCultured Whole Embryos

In order to eliminate the potentially confounding in-fluences of maternal factors, we have elected to utilizethe whole embryo culture system to initiate investiga-tions of biochemical mechanisms of chemically elicitedembryotoxicity including dysmorphogenesis. Advan-tages of this system also include a greater capacity tocontrol the concentrations of chemicals and/or their me-tabolites that actually reach the various tissues of theconceptus and the ability to stage the embryos accu-rately prior to administration of the chemicals. Disad-vantages include a lack of capacity to determine theextent to which observed toxicities would persist be-yond the period of culturing and the effort involved inthe performance of rigorous embryo-culture experi-ments.With respect to redox cyclers and chemicals that may

be converted to such, we have initiated investigationson several prototypic examples of various classes ofthese agents. These include doxorubicin (adriamycin),menadione, and paraquat as representative of quinones,diethylstilbestrol as representative of a proquinone orchemical which must be first converted metabolically toa quinone, nifuroxime, furazolidone, nitrofurazone, ni-trofurantoin, niridazole, 2-nitroimidazole, ronidazole,metronidazole, 4-nitroimidazole, and 4-methylniridazoleas representative of nitroheterocycles, chloramphenicolas a nitroarene, nitrosofluorene as an aromatic nitrosocompound and 2-acetylaminofluorene which is capableof undergoing conversion to several different types ofreactive intermediates including redox cyclers. Some ofthe results obtained have been quite surprising, othersenigmatic, and still others have been those expected.

133

JUCHAU ET AL.

Table 1. Examples of foreign organic chemicals which behave as a redox cyclers in biological systems and their reportedteratogenicity.

Chemical Teratogenic effects Species ReferencesQuinones

Doxorubicin Multiple Rats (specific) (18)Mitomycin C Skeletal Rats, mice (17)Streptonigrin Eye, CNS, trunk, tail, limbs, etc. Rats (17)Paraquat Costal cartilage defects Rats, hens (17)

Aromatic nitro compoundsNitrofurazone Limbs, digits, tail Mice (19)Nitrofurantoin Not defined Mice (20)Misonidazole Limbs, eye, brain, palate Mice (21)Nitrofen Eye, kidney, diaphragm Rats, mice (18)Nitrobenzene Multiple Rats (18)

Azo compoundsTrypan Blue Hydrocephaly, spina bifida Several (17)Evans Blue Brain, others Rats (17)Congo Red Hydronephrosis, microophthalmia, Rats (17)

hydrocephalusAminoazobenzenes Skeletal, cleft palate Mice (18)

MiscellaneousBleomycin-Fe complex Multiple Rats, mice (18)

QuinonesInitial investigations of the effects of redox cyclers

on embryonic development in vitro began with a studyof doxorubicin (22). Somewhat surprisingly, the datagathered in the studies argued against a role for redoxcycling in the observed embryotoxic effects producedby the anthracycline antibiotic. Failure of several rad-ical scavenging agents and antioxidants to amelioratethe embryotoxic effects and a lack of an observable ef-fect of changes in 02 tension of the culture mediumprovided the principal arguments against the partici-pation of redox cycling or of radical species. Interest-ingly, inclusion of an S-9 fraction from methylcholan-threne-induced rats in the culture system markedlyexacerbated the embryotoxicity and led us to speculatethat a methylcholanthrene-inducible, P-450-dependent0-demethylase catalyzed the formation ofthe 4-hydroxyderivative which is known to exhibit greater antitumoractivity than the parent 4-methoxy compound. Seem-ingly, the embryotoxic effects produced by doxorubicinwere more closely akin to its antitumor properties(which do not appear to depend on redox cycling) thanto its reputedly redox-dependent cardiotoxic effects.Menadione, a quinone which undergoes reduction cat-alyzed by the methylcholanthrene-inducible DT-dia-phorase, did not affect the embryotoxicity of doxoru-bicin (22) nor did it exhibit embryotoxicity by itself.However, the highest concentration added to the cul-ture medium was only 2 ,uM. Therefore, the embry-otoxicity in vitro of this quinone will require furtherevaluation.Paraquat is a prototype redox cycler that is capable

of producing severe toxicity in the lungs. Like doxo-rubicin, it is a quinone but is far more water-solubleand thus diffuses across cell membranes much less read-ily. At 5 ,uM concentrations, this agent produced 100%embryolethality in 12 embryos (unpublished), but at 1,uM concentrations no effect on 10 embryos could be

detected. At concentrations above 10 ,uM it was appar-ent that paraquat could produce severe effects on thevisceral yolk sac and, in view of its high polarity, it istempting to speculate that the embryotoxic effects ob-served were due to a specific effect on yolk sac integrity.Such an effect might be analogous to that produced bytrypan blue (17), an azo dye and also a potential redoxcycler. Further studies on the mechanism of the in vitroembryo toxicity of paraquat will be of high interest.

ProquinonesDiethylstilbestrol, a synthetic diphenolic estrogen,

undergoes oxidative conversion to p-quinone andp-semiquinone metabolites via direct (presumably per-oxidative) oxidation of the p-phenolic groups (14). Con-version to o-quinone and o-semiquinone intermediatescan occur via P450-dependent oxidation at carbons ad-jacent to phenolic groups to yield catechols. The cat-echols, in turn, are then peroxidatively converted to theo-quinone and (presumably) o-semiquinone radical in-termediates (15). Both of these pathways have beenimplicated in the genotoxic and carcinogenic effects pro-duced by DES (14,15). However, evidence obtained todate in our laboratory indicates that the embryotoxiceffects of DES on cultured whole embryos are directand are neither a function of redox cycling nor bioac-tivation via other metabolic pathways (23). Preliminarystudies with the estrogen antagonist, tamoxifen, alsoindicated that the effects are independent of interac-tions with estrogen receptors. Since Z,Z-dienestrol, anonestrogenic metabolite, produced effects similar tothose observed with DES, the argument for an estrogenreceptor-independent mechanism is strengthened.Also, the similar effect of hexestrol, a synthetic non-steroidal estrogen differing from DES only in lack ofthe stilbene double bond, argues against the partici-pation of p-quinone/semiquinone intermediates. All re-sults to date tend to foster the concept that the observed

134

REDOX CYCLERS AS TERATOGENS 135

effects of DES on whole cultured embryos are directand totally independent of metabolic activation. In ad-dition, supplementation ofthe culture medium with var-ious metabolic systems has failed thus far to influencethe embryotoxic effect of DES. Therefore, as with dox-orubicin, effects on the morphologic development of cul-tured embryos appears independent of redox cycling atthis stage of the research.

NitroheterocyclesThe discovery of a highly unusual asymmetric mal-

formation produced by niridazole (24,25), an antischis-tosomal nitroheterocycle, led us to investigate a seriesof structurally related chemicals in order to gain insightsinto the possible mechanism whereby the defect is elic-ited. Studies thus far (26,27) have shown that the onlychemicals capable of producing the striking asymme-trical malformation are those bearing a reducible nitrogroup. All heterocyclic nitro compounds with redox po-tentials higher than - 0.480 (with the enigmatic excep-tion of nitrofurantoin) produced the axial asymmetrywhen cultured with whole rat embryos. A very largenumber of other chemicals that have undergone studiesin the whole embryo culture system have not exhibitedthe capacity to produce the asymmetric defect. A goodcorrelation between single electron redox potentials ofnitro groups and capacity to elicit the defect was alsoobserved. The effect was also exacerbated by reducingthe 02 tension of the culture medium (28). The resultswere highly compatible with the idea that the culturedtissues of the conceptus are capable of reducing thearomatic nitro groups to one or more reactive inter-mediates which were more directly responsible for theobserved asymmetric defect. No exogenous enzymesource was required for elicitation of the abnormalityand S-9 fractions in fact, tended to reduce the embry-otoxicity (26). Interestingly, carbon monoxide tendedto ameliorate the effect, suggesting that a heme-depen-dent reduction or reoxidation reaction was required forgeneration of the reactive species. Previous investiga-tions have shown (29,30) that flavoproteins or even free,reduced flavins can reduce aromatic nitro groups (e.g.,p-nitrobenzoate) to hydroxylamines but that reductionto the fully reduced primary amine (p-aminobenzoate)required either a hemoprotein or free hemin as a cat-alyst. Addition of N-acetylcysteine to the culture me-dium did not appear to affect the incidence or severityof defects produced (21) but lowering of intracellularlevels of GSH in tissues of the conceptus with buthion-ine-S, R-sulfoximine (unpublished) markedly exacer-bated the effect. Nevertheless, experiments designedto rigorously test the possibility that redox cycling ofvarious potential nitroheterocycle intermediates is re-sponsible for embryotoxic effects of nitroheterocycleshave not yet been performed. Exacerbation ofthe effectunder conditions of lowered 02 tension tends to argueagainst a mechanism involving the exhaustion of intra-cellular reducing equivalents because higher 02 tensionshould exhaust these equivalents more effectively. It

also argues against a mechanism involving the gener-ation of hydroxyl radicals or other reactive oxygen spe-cies (singlet oxygen, hydrogen peroxide, etc.) since lev-els of these species should be increased with increasing02 tension. A mechanism involving localized depletionof tissue 02 levels remains a possibility as does a moredirect effect of intermediate organic radicals. At pres-ent, the nitroheterocyclic compounds appear to be themost probable candidates as examples of chemicals ca-pable of producing embryotoxic effects via redox cy-cling.

Aromatic NitrosoSeveral metabolites of 2-acetylaminofluorene (AAF)

have been investigated as embryotoxins in whole em-bryo cultures (6,31-33). A number of metabolites ofAAF have the potential to behave as redox cyclers al-though this mechanism has not been seriously consid-ered in terms of its carcinogenicity or mutagenicity.Nitrosofluorene, an AAF metabolite, is a relatively po-tent, direct-acting dysmorphogenic agent in vitro andthe possibility of its conversion to redox cycling radicalintermediates is conceivable. Alterations of extracel-lular as well as intracellular GSH levels also markedlyinfluences its dysmorphogenic effects (34). Again, how-ever, much more investigation will be required to de-termine whether redox cycling can be implicated evenpartially. Certainly many other potential mechanismsare conceivable.

REFERENCES

1. Juchau, M. R. The Biochemical Basis of Chemical Teratogenesis.Elsevier/North-Holland, New York, 1981.

2. Workshop in Teratology. In vitro teratogenesis testing. Teratog.Carcinog. Mutag. 2: 221-361 (1982).

3. Fantel, A. G., Greenaway, J. C., Juchau, M. R., and Shepard,T. H. Teratogenic bioactivation ofcyclophosphamide in vitro. LifeSci. 25: 67-72 (1979).

4. Fantel, A. G., Greenaway, J. C., Shepard, T. H., and Juchau,M. R. The teratogenicity of cytochalasin D and its inhibition bydrug metabolism. Teratology 23: 223-233 (1981).

5. Greenaway, J. C., Fantel, A. G., Shepard, T. H., and Juchau,M. R. The in vitro teratogenicity of cyclophosphamide in ratembryos. Teratology 25: 335-344 (1982).

6. Faustman-Watts, E., Greenaway, J. C., Namkung, M. J., Fantel,A. G., and Juchau, M. R. Teratogenicity in vitro of 2-acetylami-nofluorene: role of biotransformation. Teratology 27: 19-29 (1983).

7. Juchau, M. R., Giachelli, C. M., Fantel, A. G., Greenaway, J.C., Shepard, T. H., and Faustman-Watts, E. M. Effects of 3-methylcholanthrene and phenobarbital on the capacity ofembryosto bioactivate teratogens during organogenesis. Toxicol. Appl.Pharmacol. 80: 137-147 (1985).

8. Juchau, M. R., Bark, D. H., Shewey, L. M., and Greenaway, J.C. Generation of reactive dysmorphogenic intermediates by ratembryos in culture: effects of cytochrome P-450 inducers. Toxicol.Appl. Pharmacol. 81: 533-544 (1985).

9. Faustman-Watts, E. M., Giachelli, C. M., and Juchau, M. R.Carbon monoxide inhibits embryonic monooxygenation and em-bryotoxic effects of proteratogens in vitro. Toxicol. Appl. Phar-macol. 83: 590-596 (1986).

10. Kappus, H. Overview of enzyme systems involved in bio-reduc-tion of drugs and in redox cycling. Biochem. Pharmacol. 35: 1-7(1986).

11. Reed, D. J. Cellular defense mechanisms against reactive me-

136 JUCHAU ET AL.

tabolites. In: Bioactivation of Foreign Compounds (M. W. An-ders, Ed.), Academic Press, New York, 1985, pp. 71-108.

12. Lloyd, D. Chairman's Summary of Session B. Biochem. Phar-macol. 35: 65 (1986).

13. Wefers, H., and Sies, H. Generation of photoemissive speciesduring quinone redox cycling. Biochem. Pharmacol. 35: 22-24(1986).

14. Metzler, M. Mechanisms of carcinogenesis induced by diethyl-stilbestrol. In: Comparative Perinatal Carcinogenesis (H. M.Schuller, Ed.), CRC Press, Boca Raton, FL, 1984, pp. 138-150.

15. Li, S. A., Klicka, J. K., and Li, J. J. Estrogen 2- and 4-hydrox-ylase activity, catechol estrogen formation, and implications forestrogen carcinogenesis in the hamster kidney. Cancer Res. 45:181-186 (1985).

16. Liehr, J. G. Possible role of 4', 4"- diethylstilbestrol quinone indiethylstilbestrol carcinogenesis. J. Toxicol. Environ. Health 16:693-701 (1985).

17. Shepard, T. H. Catalog of Teratogenic Agents, 4th ed. JohnsHopkins Press, Baltimore, 1983, pp. 440-443.

18. Schardein, J. L. Chemically Induced Birth Defects. Marcel Dek-ker, New York, 1985.

19. Nomura, T., Kimura, S., Isa, Y., Tanaka, H., and Sakamoto, Y.Teratogenic and carcinogenic effects of nitrofurazone on themouse embryo and newborn. Teratology 12: 206-207 (1975).

20. Nomura, T., Kimura, S., Isa, Y., Tanaka, H., and Sakamoto, Y.Teratogenic effects of some antimicrobial agents on mouse em-bryo. Teratology 14: 250 (1976).

21. Michel, C., and Fritz-Niggli, H. Teratogenic and radiosensitizingeffects of misonidazole on mouse embryos. Brit. J. Radiol. 54:154-155 (1981).

22. Fantel, A. G., Greenaway, J. C., and Juchau, M. R. The embry-otoxicity of adriamycin in rat embryos in vitro. Toxicol. Appl.Pharmacol. 80: 155-165 (1985).

23. Beyer, B. K., Greenaway, J. C., and Juchau, M. R. DES-inducedembryotoxicity in vitro. Teratology 32: 45A (1985).

24. Fantel, A. G., Greenaway, J. C., and Juchau, M. R. Teratogenicmetabolism of niridazole. Teratology 29: 27A (1984).

25. Fantel, A. F., Greenaway, J. C., and Juchau, M. R. Extraem-

bryonic bioinactivation of niridazole teratogenicity in vitro. Ter-atology 32: 37A (1985).

26. Fantel, A. G., Greenaway, J. C., Walker, E., and Juchau, M. R.The toxicity of niridazole in rat embryos in vitro. Teratology 33:105-112 (1986).

27. Greenaway, J. C., Fantel, A. G., and Juchau, M. R. On thecapacity of nitroheterocyclic compounds to elicit an unusual axialasymmetry in cultured rat embryos. Toxicol. Appl. Pharmacol.82: 307-316 (1986).

28. Greenaway, J. C., Mirkes, P. E., Walker, E. A., Juchau, M. R.,Shepard, T. H., and Fantel, A. G. The effect of oxygen concen-tration on the teratogenicity of salicylate, niridazole, cyclophos-phamide and phosphoramide mustard in rat embryos in vitro.Teratology 32: 287-295 (1985).

29. Juchau, M. R., Krasner, J., and Yaffe, S. J. Model systems foraromatic nitro group reduction: relationships to tissue-catalyzedreactions. Biochem. Pharmacol. 17: 443-455 (1970).

30. Symms, K. G., and Juchau, M. R. The aniline hydroxylase andnitroreductase activities of partially purified cytochromes P-450and P-420 and cytochrome b5 solubilized from rabbit hepatic mi-crosomes. Drug Metab. Disp. 2: 194-201 (1974).

31. Faustman-Watts, E. M., Yang, H. Y. L., Namkung, M. J.,Greenaway, J. C., Fantel, A. G., and Juchau, M. R. Mutagenic,cytotoxic and teratogenic effects of 2-acetylaminofluorene and re-active metabolites in vitro. Terat. Carcin. Mutag. 4: 273-283(1984).

32. Faustman-Watts, E. M., Greenaway, J. C., Namkung, M. J.,Fantel, A. G., and Juchau, M. R. Teratogenicity in vitro of twodeacetylated metabolites of N-hydroxy-2-acetylaminofluorene.Toxicol. Appl. Pharmacol. 76: 161-172 (1984).

33. Faustman-Watts, E. M., Namkung, M. J., Greenaway, J. C., andJuchau, M. R. Analyses of metabolites of 2-acetylaminofluorenegenerated in an embryo culture system: relationship of biotrans-formation to teratogenicity in vitro. Biochem. Pharmacol. 34:2953-2960 (1985).

34. Harris, C., Namkung, M. J., Fantel, A. G., and Juchau, M. R.The regulation of glutathione in rat embryos and visceral yolksacs in the embryo culture system and its effects on nitrosoflu-orene-induced malformations. Toxicologist 6: 97 (1986).