Methanol metabolism and embryotoxicity in rat and mouse conceptuses: comparisons of alcohol...

Reproductive Toxicology 17 (2003) 349–357

Methanol metabolism and embryotoxicity in rat and mouse conceptuses:comparisons of alcohol dehydrogenase (ADH1), formaldehyde

dehydrogenase (ADH3), and catalase

Craig Harris∗, Show-Won Wang, Juan J. Lauchu, Jason M. HansenToxicology Program, Department of Environmental Health Sciences, School of Public Health, University of Michigan,

1420 Washington Heights, Ann Arbor, MI 48109-2029, USA

Received 20 January 2003; received in revised form 20 January 2003; accepted 11 February 2003

Abstract

Mouse embryos are more sensitive than rat embryos in response to methanol (CH3OH) and its ability to elicit developmental abnor-malities. Intrinsic differences in the metabolism of CH3OH to formaldehyde (HCHO) and formic acid (HCOOH) by the enzymes alcoholdehydrogenase (ADH1), formaldehyde dehydrogenase (ADH3), and catalase may contribute to the observed species sensitivity. Specificactivities for enzymes involved in CH3OH metabolism were determined in rat and mouse conceptuses during the organogenesis period of8–25 somites. Spatial activity relationships were also compared separately in heads, hearts, trunks, and the visceral yolk sac (VYS) fromearly (7–12 somites) and late (20–22 somites) organogenesis-stage rat and mouse embryos. Catalase activities were similar between ratand mouse conceptuses. In the mouse heart, catalase activities were consistently lower when compared to other tissues. Specific activitiesfor catalase were consistently highest in the VYS of both species when compared to other tissues of the embryo. These activities werehighly significant in the 6–12 somite VYS. ADH1 activities were significantly higher in embryos when compared to VYS in both species,except for a 27% lower activity in the early 8–10 somite mouse embryo. Mouse ADH1 activities in the VYS were significantly lowerthroughout the organogenesis period when compared to the rat VYS or embryos of either species. Mouse activities were lower overall inspecific tissues of the embryo but maintained the same relative proportions as in the rat. ADH3 activities in the rat VYS were significantlyhigher by 20% than those in the mouse. Mouse embryo ADH3 activities were slow to mature, starting at a level 42% below rat, and failedto reach optimal levels until the 14–16-somite stage. Heart ADH3 activities were also significantly lower in the mouse embryo at the7–12-somite stage. Both species have lower ADH3 activities in the early heart, relative to other embryonic tissues. These results show amore slowly maturing capacity of the mouse embryo to remove HCHO, which provides a rationale for increased sensitivity of this speciesto CH3OH-induced embryotoxicity and teratogenicity.© 2003 Elsevier Science Inc. All rights reserved.

Keywords: Methanol; Mouse; Rat; Embryo; Visceral yolk sac; Alcohol dehydrogenase1; Alcohol dehydrogenase3; Catalase

1. Introduction

Methanol (CH3OH) is a widely used industrial solventthat has also been proposed as an alternative motor fuel in theUnited States. If used for this purpose, it could contribute toan increased risk for toxicity due to greater human exposureto CH3OH vapor. The probable incidence of malformationsand mechanisms of CH3OH-related terata in humans are notwell defined. Studies utilizing rodent models have describedCH3OH embryotoxicity following different routes of expo-sure. Inhalation of CH3OH (10,000–15,000 ppm 7 h/day on1 or 2 consecutive days) in pregnant CD-1 mice-produced

∗ Corresponding author. Tel.:+1-734-9363397; fax:+1-734-7638095.E-mail address: [email protected] (C. Harris).

embryos with numerous defects in a dose-dependent man-ner, most notably exencephaly, cleft palate, reduced fetalweight, and supernumerary cervical ribs. An increase inembryo/fetal death was also observed[1–4]. Rat embry-otoxicity (Sprague–Dawley) has also been reported byNelson et al.[5] following high-dose inhalation exposure.Similarly, direct exposure of rodent conceptuses to CH3OHin whole embryo culture caused exencephaly, cleft palate,cervical skeletal defects, reduced body weight, increasedembryo/fetal death, decreased somite number, smaller headlength, and lower developmental scores[6,7]. Comparisonbetween CH3OH exposures in rat (Sprague–Dawley) andmouse (CD-1) indicated that the mouse was more sensitiveto CH3OH, as evidenced by a greater incidence and severityof teratogenesis and embryotoxicity as compared to the rat

0890-6238/03/$ – see front matter © 2003 Elsevier Science Inc. All rights reserved.doi:10.1016/S0890-6238(03)00013-3

350 C. Harris et al. / Reproductive Toxicology 17 (2003) 349–357

[6–8]. Currently, the basis for species specificity of CH3OHexposure is not understood but may involve differences inthe major CH3OH detoxication pathways, specifically alco-hol dehydrogenase (ADH1), formaldehyde dehydrogenase(ADH3), and catalase.

Initially, CH3OH is metabolized through one of atleast three different pathways. The first and second in-volve a common intermediate, hydrogen peroxide (H2O2).The H2O2 intermediate may be generated as a result ofNADPH-dependent microsomal electron transfer and cancontribute to the direct oxidation of CH3OH by the actionof catalase (Reaction 1a) or may undergo the Fenton reac-tion to produce hydroxyl radicals (•OH), which in turn reactspontaneously with CH3OH to yield formaldehyde (HCHO;Reaction 1b)[7,9,10]. The third significant pathway forCH3OH metabolism involves the alcohol dehydrogenase(ADH1) pathway (Reaction 1c)[11].

Reaction 1a : CH3OH + H2O2catalase→ HCHO

Reaction 1b : H2O2 → •OHCH3OH + •OH → HCHO

Reaction 1c : CH3OH + NAD+ ↔ HCHO+ NADH

In the mitochondria, HCHO is further metabolized toformic acid (HCOOH) by the action of alcohol dehydroge-nase2 (ADH2), but in the cytosol, it is converted to HCOOHby a series of reactions involving glutathione-dependentformaldehyde dehydrogenase (ADH3) (Reactions 2–4),which is identical to alcohol dehydrogenase3[12], and isfunctionally related to aldehyde dehydrogenase1 (ADH1)[13].

Reaction 2 : HCHO+ GSH↔ S-hydroxylmethyl GSH

(spontaneous)

Reaction 3 : S-hydroxylmethyl GSH+ NAD+

↔ S-formyl GSH(via ADH3/ADH1)

Reaction 4 : S-formyl GSH

↔ GSH+ HCOOH

(viaS-formyl GSH hydrolase)

Methanol-related ocular toxicity has been attributed tothe accumulation of HCOOH[7,14–16], which is believedto contribute to the human clinical manifestations of blind-ness through inhibition of terminal cytochrome oxidases ofthe mitochondrial respiratory chain. Formic acid exposurein vitro also causes rat and mouse embryonic malforma-tions, and these have been suggested to be largely a resultof changes in pH[8]. Still, it is not clear whether CH3OH,HCHO, or HCOOH are directly responsible for teratogene-sis and/or embryolethality in rodent models.

To better understand the potential mechanistic pathwaysinvolved in CH3OH teratogenesis, assays determining spe-cific activities of enzymes involved in CH3OH metabolism,specifically ADH1, ADH3, and catalase were performed in

rat and mouse conceptuses of similar developmental stage(based on somite number). Temporal and/or spatial differ-ences in the activities of these enzymes could contribute tomechanisms underlying the observed differences in sensi-tivity and resistance to CH3OH in mouse and rat concep-tuses, respectively. Moreover, specific tissues were analyzedto provide a better understanding of possible CH3OH tar-gets in early (7–12 somites) and late stage (20–22 somites)embryos. The whole embryo culture system was employedto facilitate comparisons between conceptuses of differentspecies and specific developmental stages, thus improvingthe fidelity of the species and tissue comparisons.

2. Materials and methods

2.1. Chemicals

Hanks’ balanced salt solution (HBSS) was purchasedfrom Gibco BRL (Grand Island, NY). Pyridine nucleotides(NAD+), reduced glutathione (GSH), glycine, semicar-bazine hydrochloride, Tyrode’s salts, and 4-amino-3-hydra-zino-5-mercapto-1,2,4-triazole (Purpald) were purchasedfrom Sigma Chemical Co. (St. Louis, MO). Sodium py-rophosphate and potassium phosphate were obtained fromJ.T. Baker Inc. (Phillipsburg, NJ). Ethanol was from Mid-west Grain Products Co. (Weston, MI). EDTA, CH3OH,and HCHO were from Mallinckrodt Inc. (Paris, Kentucky).

2.2. Animals

Time-mated primigravida Sprague–Dawley rats or CD-1mice were obtained on day 9 and day 7 of gestation, re-spectively, from the University of Michigan ReproductiveSciences Program, P-30 Small Animal Core facility. Themorning following copulation, a vaginal smear was usedto confirm pregnancy. This sperm-positive vaginal smearwas designated as day 0 of gestation. The animals weremaintained in a 10 h/14 h light/dark cycle. Food and waterwere provided ad libitum until pregnant rat and mouse ex-plantation on day 10 (GD10) and day 8 (GD8) of gestation,respectively.

2.3. Whole embryo culture

Whole embryo cultures have been used throughout instudies designed to evaluate the embryotoxic potential ofCH3OH and its metabolites because of the ability to strictlycontrol dose, duration of exposure, and growth conditions.We have chosen to use the same system to generate andcompare enzyme ontogeny profiles for enzymes involved inmethanol because of the ability to directly correlate to otherin vitro studies and to provide the most direct means of com-paring stage-specific activities between the rat and mousespecies.

C. Harris et al. / Reproductive Toxicology 17 (2003) 349–357 351

The pregnant dams were anesthetized with ether and theconceptuses were explanted using explantation protocols aspreviously described[17]. The rat and mouse conceptuseswere placed in separate culture bottles containing either 33%rat serum in HBSS (pH= 7.4) or 75% rat serum in Tyrode’ssalt solution (TSS; pH= 7.4), respectively, supplementedwith penicillin G (100 IU/ml) and streptomycin (50 IU/ml).One conceptus per milliliter was used in a total volume of10–15 ml of medium. The medium was warmed to 37◦C andgassed with 20% O2/5% CO2/75% N2 at the beginning ofthe culture period (20 h). The culture bottles were placed ina roller incubator overnight and regassed with 95% O2/5%CO2 on the following morning for the final 4 h of incu-bation. The conceptuses were dissected using a dissectionmicroscope where the Reichert’s membranes and deciduawere removed. Tissues such as visceral yolk sacs (VYS) andembryos were placed in 0.05 M potassium phosphate buffer(pH = 7.0) and stored for enzyme assays at−75◦C. Addi-tionally, some embryos were designated for tissue dissectioninto heads, hearts, and trunks and were stored for enzymeassays at−75◦C.

2.4. Protein assay

Protein content was determined by the method of Bradford[18] and modified for 96-well microtiter plate as describedin Harris et al.[19]. Bovine gamma globulin was used as astandard.

2.5. Catalase assay

Tissues were thawed, ultrasonicated, and utilized im-mediately for the determination of catalase activity by the

Fig. 1. Catalase-specific activities in embryos and visceral yolk sacs of rats and mice from 6 to 28 somites. Data are represented as nmoles HCHOformed per min per milligram protein, mean+ S.E.M.

method previously described by Johansson and Borg[20].The enzymatic reaction was initiated by adding 100�l ofa catalase-containing sample. Samples were added into themixture containing 110�l of 25 mM KH2PO4–NaOH (pH7.0) with 50�l CH3OH and 10�l H2O2. This mixturewas mixed and placed in a shaking incubator for 20 min atroom temperature. The enzymatic reaction was terminatedby the addition of 50�l of 7.8 M potassium hydroxide toeach tube. The addition of 100�l of 34.2 mM Purpald in480 mM hydrochloric acid was made immediately to detectHCHO formed and was followed by a second incubationwith continuous shaking for 10 min at room temperatureto complete the reaction. The products of the reaction be-tween HCHO and Purpald were oxidized by adding 50�lof 65.2 mM potassium peroxide in 470 mM potassium hy-droxide to each tube. Following centrifugation at 9500× g

for 10 min, the absorbance of 150�l of supernant wasmeasured at 550 nm in a microplate reader. Sample con-centrations of HCHO were determined by comparison toan authentic standard curve and catalase-specific activitieswere expressed as nmol HCHO formed/min/mg protein.

2.6. ADH1 assay

Thawed tissues were ultrasonically disrupted and utilizedimmediately for the determination of enzyme activity, usingthe method described by Wilson et al.[21]. ADH1 activitywas determined by the addition of 50�l of tissue ho-mogenates to 150�l of reaction mixture containing 1.8 mMNAD+, 1.0 mM GSH, 6.2 mM semicarbazide hydrochlo-ride, 19.1 mM glycine, and 85.5 mM sodium pyrophosphate(pH 8.0). The reaction was started by addition of 9�l


ethanol, mixed, and incubated at room temperature for5 min. The ADH1 activity was determined by the formationof NADH which was determined using a Beckman DU-650spectrophotometer (ε = 6220 M−1 cm−1 at 340 nm). Spe-cific activity was defined as pmol NADH formed/min/mgprotein.

2.7. Formaldehyde dehydrogenase (ADH3) assay

Slight modification of original method as described byUotila and Koivusalo[22] was used. ADH3 activity wasdetermined by the addition of 50�l of tissue homogenatesto the 150�l of reaction mixture containing 0.12 M sodium

Fig. 2. Catalase-specific activities in mouse (A) and rat (B) heads, hearts, trunks, and visceral yolk sacs of varying developmental stages (6–12, 13–20,and 21–28 somites). Data are represented as nmoles HCHO formed per min per milligram protein, mean+ S.E.M. ∗Statistically significant differencebetween like tissues of each species at similar developmental stages.

phosphate (pH 8.5), 1 mM EDTA, 3 mM pyrazole (to blockthe reaction of HCHO to CH3OH), 0.1 M GSH, and 0.04 MNAD+. The reaction was started by addition of 10�l HCHO,mixed, and incubated at room temperature. ADH3 activ-ity was measured after 5 min by a Beckman DU-650 spec-trophotometer (ε = 6220 M−1 cm−1 at 340 nm). Specificactivity was defined as pmol NADH formed/min under theforward assay condition/mg protein.

2.8. Statistical analysis

One-way analysis of variance (ANOVA) followed bya Tukey’s post hoc test was used to determine overall


differences between mean specific activities. Comparisonsof activity levels between each somite range of the twospecies were made using Student’st-test. Statistical signifi-cance was determined atP = 0.05.

3. Results

3.1. Catalase

Embryo and tissue samples for catalase assay were col-lected over wider somite ranges than used for ADH1 andADH3 due to a less sensitive assay procedure and the needto pool greater amounts of tissue for adequate detection.Catalase-specific activities increased as organogenesis pro-ceeded in both rat and mouse conceptuses. Specific activitiesfor the generation of HCHO from CH3OH ranged from 1.5to 17.0 nmol HCHO/min/mg protein and did not differ sig-nificantly between embryos and VYSs of the rat and mouse(Fig. 1). Comparisons between specific conceptal tissuesfrom the two species also showed few significant differencesin activities (Fig. 2A and B). Specific activities in the VYSwere consistently higher than other conceptal tissues inde-pendent of developmental stage or species. Catalase-specificactivity in rat heart was found to be greater than two-foldhigher than in mouse heart at the 6–12-somite stage. Cata-lase activity in other tissues did not show a significant dif-ference between the species at any somite stage.

3.2. ADH1

Specific activities for ADH1 in rat and mouse embryoswere similar between the 11–13- and 23–25-somite ranges,

Fig. 3. ADH1-specific activity ontogeny for rat and mouse visceral yolk sacs and embryos from 8-somites to 25-somites. Data are represented aspmoles NADH formed per min per milligram protein, mean+ S.E.M. ∗Statistically significant difference between like tissues of each species at similardevelopmental stages.

ranging from 240 to 490 pmol NADH/min/mg protein.ADH1-specific activities were significantly lower by 25%in the mouse embryo at the early 8–10-somite stage (Fig. 3).Visceral yolk sac ADH1 activity in both the mouse andrat showed very similar developmental activity profiles(Fig. 3), although rat VYS ADH1 activities were 15–25%higher than those seen in the mouse. Specific activitiesconverged at the 23–25-somite stage embryo and were nolonger significantly different. Evaluation of activities inselected tissues (heads, hearts and trunks) at early[7–12]and later[20–22]somite ranges were consistent with wholeembryo measurements showing similar patterns of relativeactivity. Mouse ADH1-specific activities were lower thanrat at 7–12 somites (Fig. 4A). As in the whole embryo,activities converged at the 20–22-somite stage and wereno longer different (Fig. 4B). Subsequent measurements ofembryonic ADH1 activity showed similar ADH1 activitybetween species from 11–13 to 23–25 somite embryos.

3.3. Formaldehyde dehydrogenase

Both rat and mouse VYS ADH3-specific activities re-mained relatively consistent from early-stage embryos (8–10somites) to late-stage embryos (23–25 somites) with spe-cific activities ranging from 300–675 pmol NADH/min/mgprotein (Fig. 5). However, comparisons between species in-dicate that the rat VYS contained significantly increasedADH3 activity, approximating a 15–20% difference through-out each somite range during this developmental period.

Much like the VYS, the rat embryo maintained uniformADH3 activities from the early to late stages of develop-ment and showed no statistically significant differences overtime (Fig. 5). The mouse, however, had significantly lower


Fig. 4. ADH1-specific activities of heads, hearts, and trunks from early(A) and late (B) embryos. Data are represented as pmoles NADH formedper min per milligram protein, mean+ S.E.M. ∗Statistically significantdifference between like tissues of each species at similar developmentalstages.

ADH3 activities by 42% in the 8–10 somite and 11–13somite embryos and did not reach optimal levels until the14–16-somite stage, after which no difference from the ratwas seen.

Comparison of embryonic tissues showed that only heartADH3 activity was different between species in young em-bryos (7–12 somites), at which point the mouse heart con-tained only 30% of the ADH3 activity that was measuredin the rat heart (Fig. 6A). Other tissues at this stage werenot significantly different between species. In older embryos(20–22 somites), there were no significant species differ-ences between any embryonic tissues (Fig. 6B).

4. Discussion

Conflicting hypotheses have been presented in the liter-ature regarding the role of metabolism in mechanisms ofCH3OH toxicity. Andrews et al.[7] concluded that the par-ent CH3OH was the ultimate embryotoxicant because theywere unable to detect significant changes in concentrationsdissolved in the culture media over a 24-h period. Thisobservation does not preclude the possibility of metabolicgeneration of small quantities of very reactive specieswithin sensitive cells of the embryo proper. This fact issupported by later studies from the same group that showsignificant increases in14C-methanol incorporation intoDNA and proteins in embryonic tissues following the sameCH3OH exposures that could lead to alterations of proteinfunction or gene expression[23]. No species comparisonswere made using these endpoints, although enzyme activitydifferences involved in DNA methylation and conversionof CH3OH to intermediates capable of binding to proteinsmay offer some clue to differences in sensitivity.

A number of investigators have concluded that HCHO isan important primary metabolite of CH3OH and we haveshown that HCHO, added directly to the culture medium, canelicit embryotoxicity at concentrations 1000-fold lower thanare required to produce the same degree of toxicity follow-ing direct CH3OH exposure[24]. This finding suggests thatmetabolism of a relatively small percentage of the CH3OHto HCHO within the conceptus could also be the cause of ob-served acute embryotoxicity. Based on the Andrews et al.’s[7] data and other studies[25], it is possible that a signif-icant metabolism of CH3OH to the secondary metabolite,HCOOH, occurs in the rodent, but its efficient metabolic re-moval prevents concentrations from increasing to levels suf-ficient to produce acidosis. Toxicity due to acidosis resultingfrom HCOOH accumulation is of concern in primates andin rodents where the HCOOH removal processes are inhib-ited [26,27]. Inhalation of CH3OH (10,000 ppm for 6 h) hasbeen shown to cause exencephaly, a commonly describedmalformation in CH3OH-treated embryos[28]. Direct expo-sure to formate via oral gavage (750 mg/kg) did not producean increased concentration of formate in embryonic tissues(2.2 mM) but rather produced formate concentrations simi-lar to those following CH3OH inhalation at 15,000 ppm for6 h (2.0 mM). However, while inhalation of CH3OH wassufficient to elicit exenchephaly, direct exposure to formatedid not result in malformation. Similarities in formate con-centrations in CH3OH-exposed embryos and the absenceof malformation in sodium formate treated embryos sug-gest that formate is not responsible for CH3OH-inducedexencephaly and possibly not to other related terata as well.

More recent investigations into mechanisms of methanoltoxicity also suggest that the generation of free radicals andinitiation of lipid peroxidation may contribute to embryotox-icity [10], although these mechanisms have not yet been sys-tematically evaluated in rodent embryos. The studies citedabove provide some insight into possible mechanisms of


Fig. 5. ADH3-specific activity ontogeny for rat and mouse visceral yolk sacs and embryos from 8-somites to 25-somites. Data are represented aspmoles NADH formed per min per milligram protein, mean+ S.E.M. ∗Statistically significant difference between like tissues of each species at similardevelopmental stage.

CH3OH toxicity, but do little to address the basis for speciessensitivity. If the biotransformation of CH3OH by conceptalenzymes proves necessary for causing embryotoxicity andteratogenicity in rodents, spatial and temporal differences inthe activities of enzymes that convert CH3OH to more reac-tive chemical species such as HCHO and the compromisedconversion to HCOOH could also contribute to the speciessensitivities observed between mice and rats. Overall man-ifestations of toxicity depend on the balance between therate of generation of reactive intermediates and the pathwaysresponsible for further metabolism and detoxication of thebioactivated product.

The spatial activity distinctions between embryo andVYS are important because the rat and mouse inverted VYScompletely encompass the embryo to form a physical andmetabolic barrier through which a chemical must pass be-fore reaching the embryo proper. Significant bioactivationand detoxication activity in the VYS could result in very lit-tle toxicant actually reaching the embryo. In rat and mousecomparisons between ADH1, ADH3, and catalase activities,differences emerge that could help explain the increasedsensitivity of the mouse to CH3OH. Enzyme ontogenyprofiles for catalase show little difference between the twospecies (rat and mouse) that could explain selective speciesembryotoxicity and would, therefore, not be expected tocontribute to a increase of HCHO in the mouse. The sim-ilarities of catalase activity were seen in both embryo andVYS. Mouse VYS has significantly lower specific activitiesfor both ADH1 and ADH3, compared to the rat, indicatingthat at a similar dose of CH3OH, less is being converted to

HCOOH and more CH3OH may be reaching the embryoproper. Within the embryo proper, optimal rat and mouseADH1 and ADH3 activities are seen from the 14–25-somiteranges and are virtually identical. These activities differsignificantly, however, in the earlier stages where the mouseADH1 activities are lower at the 8–10-somite stage andADH3 activities are significantly lower through the 8–10-and 11–13-somite stages. These findings indicate a delayin the maturation of CH3OH detoxication enzyme activitiesin the mouse during the most sensitive developmental stagewhere many anatomic defects are believed to be elicited.Significantly lower ADH3 activities would suggest an in-ability to rapidly remove HCHO and would be expected tocontribute to greater embryonic macromolecule binding asseen in the14C-incorporation studies described above[23].

The activity of ADH3 requires glutathione (GSH) as a co-factor to transform HCHO to HCOOH. Glutathione is usu-ally present in high concentrations in embryonic cells andis involved in the detoxification of numerous reactive chem-icals capable of causing embryotoxicity[29–32]. WithoutGSH, the transformation of CH3OH to HCOOH could notoccur via ADH3. Mouse embryonic and VYS GSH con-centrations are approximately 11–15 pmol GSH/�g proteinand 20–23 pmol/�g protein, respectively ([33]; Harris, un-published data). Rat embryos generally contain significantlymore GSH (>30–45%) with embryos measuring 15–25 pmolGSH/�g protein and VYSs in the 25–30 pmol/�g proteinranges[34]. Greater GSH concentrations in the rat may pro-vide a more responsive antioxidant defense system againstCH3OH and its metabolites. Higher levels of GSH also


Fig. 6. ADH3-specific activities of heads, hearts, and trunks from early(A) and late (B) embryos. Data are represented as pmoles NADH formedper min per milligram protein, mean+ S.E.M. ∗Statistically significantdifference between like tissues of each species at similar developmentalstage.

provide more cofactor for the GSH-dependent ADH3 to fa-cilitate transformation of the highly reactive HCHO, to theless harmful formic acid.

The confirmation of whether CH3OH toxicity is due to theparent compound itself or a subsequently formed metabolite,such as HCHO or HCOOH, has yet to be made unequiv-ocally. Several hypotheses have been presented to suggestthat the ultimate toxicant resulting from CH3OH exposure insome tissues is formic acid. Andrews et al.[7] reported thatsodium formate and HCOOH are toxic to embryos exposeddirectly in whole embryo culture. Although the mechanismis unknown, formate and HCOOH toxicity is suggested tobe a result of acidosis and changes in intracellular pH. In

culture, HCOOH or formate is shown to be 6–12-fold morepotent than CH3OH, as supported by the data of Andrewset al.[8], but accumulation to concentrations needed to initi-ate teratogenesis are unlikely to be reached in vivo followingCH3OH exposures. Following CH3OH exposure, HCOOHand sodium formate concentrations are not detectable[7],suggesting that the other metabolites, such as HCHO mayalso be responsible for teratogenicity.

Few data are available to confirm a role for HCHO inCH3OH embryotoxicity, mostly due to the short half-life ofHCHO in biologic fluids and our compromised ability to ac-curately monitor its production within cells. Formaldehydeis very reactive and was found to be approximately 1000-foldmore potent than CH3OH in eliciting toxicity in conceptusesthat were exposed directly in whole embryo culture (Hansenet al., manuscript in preparation). Due to its reactive nature,HCHO is an excellent candidate for the CH3OH metabolitemost responsible for CH3OH-related embryotoxicity as onlysmall concentrations would be necessary to illicit deleteriouseffects. This role for HCHO would imply that ADH3 activi-ties are very important in embryonic protection and suggestthat it is the inability to detoxicate HCHO, rather than di-rect effects of CH3OH or HCOOH, that occurs in CH3OHembryotoxicity.

Specific activities for the three enzymes in microdis-sected embryonic tissues (head, hearts, and trunks) showa relatively high degree of variability during the earlierstages of organogenesis but activities tend to converge asdevelopment proceeds. Heart catalase and ADH3 activitieslag behind head and trunk in nearly every case studied,with the mouse activities being lower than rat at the earlieststages of organogenesis. No attempts have yet been madeto correlate activities in specific embryonic cells or tissueswith specific abnormalities. It is reasonable to expect thatcell and tissue-specific variations in enzyme activity couldcontribute selective lesions.

Enzyme ontogenies have been helpful in the determina-tion of susceptibility to toxicants during development andbetween sensitive and resistant species[35,36]. Similarly,catalase, ADH1, and ADH3 ontogenies provide valuableinsight as to which metabolite is most likely responsible forCH3OH embryotoxicity. Our observations in these studiesprovide a rationale for species- and tissue-selective CH3OHtoxicity, suggesting that HCHO may be the most likely de-velopmental toxicant as compared to CH3OH or HCOOH.Further study is required to fully understand the genera-tion of HCHO and how it interacts in both mouse and ratconceptuses.

Acknowledgments

We thank Sara Jane Larsen, Rita Berberian, Dr. Chung-Liang Kuo, Suree Jianmongkol, and Malia Dixon for theirtechnical assistance and constructive suggestions during theresearch.


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Methanol metabolism and embryotoxicity in rat and mouse conceptuses: comparisons of alcohol...

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