TERATOLOGY 44:251-257 (1991)
The Effect of In Vivo Glutathione Depletion With Buthionine Sulfoximine on Rat Embryo Development BARBARA F. HALES AND HEATHER BROWN Department of Pharmacology and Therapeutics, McGill University, 3655 Drummond Street, Montrt!al, Quebec, Canada H3G 1 Y6
ABSTRACT
Glutathione is a n abundant endogenous nucleophile whose depletion may have adverse effects on a number of cellular processes, including protein and DNA synthesis, amino acid transport, and detoxification of reactive electrophiles. Previous studies have indicated that a certain basal level of glutathione is essential for normal development in vitro in the whole rat embryo culture system. The objective of this study was to determine the effect of glutathione depletion with buthionine sulfoximine on the development of rat embryos in vivo. Timed pregnant Sprague-Dawley rats were treated by gavage on days 10 and 11 of gestation with saline (control) or with L-buthionine-(S,R)sulfoximine (4or 8 mmol/kg). Pregnancy outcome was assessed on day 12 or day 20 of gestation. The glutathione content of embryos and of maternal organs was measured on day 12. Glutathione concentrations were significantly decreased in the embryos and in maternal muscle and ovary but not in maternal liver and kidney. Glutathione depletion, when assessed on day 12 of gestation, was found to result in a n increase in the numbers of dead and malformed embryos. Treatment with the higher dose of buthionine sulfoximine resulted in the death of 13.2% of the total implanted embryos; of the surviving embryos, 21.7% were malformed. There was also a significant decrease on day 12 after treatment with the higher dose of buthionine sulfoximine in the embryonic total morphological score, a measure of the developmental stage of the embryos (control, 57.721.0; 8 mmolikg buthionine sulfoximine, 52.82 1.8).No significant effect of treatment with either dose of buthionine sulfoximine with respect to growth parameters was observed on day 12 of gestation. Interestingly, no significant effect of treatment with either dose of buthionine sulfoximine on numbers of pups, resorptions, malformations, or fetal weight was found in the litters examined on day 20 of gestation. Thus, after glutathione depletion with buthionine sulfoximine, a n increase in malformations and developmental delay is found on day 12 of gestation; however the malformations observed in day 12 embryos apparently do not persist during later development.
Glutathione is a tripeptide abundant in the cells of most organisms. This nucleophile is thought to be a n essential cell component, playing a n important role as a cellular antioxidant as well a s in amino acid transport. Increased intracellular glutathione protects the cell against the toxic effects of a variety of drugs and of radiation. In a recent report, a n increased capacity to synthesize glutathione in a model strain of E . coli was associated with enhanced resistance to y-irradiation (Moore et al., '89). Decreased glutathione synthesis or content re0 1991 WILEY-LISS,
INC.
sults in greater sensitivity to heavy metals (Singhal et al., '87; Ishikawa et al., '881, endogenous electrophiles (Kuzuya et al., '89), cancer chemotherapeutic agents, and radiation (Ishikawa et al., '89; Mitchell et al., '89). The maintenance of reduced glutathione may be particularly important dur-
Received October 10, 1990; accepted March 19. 1991. Address reprint requests to Dr. B. F. Hales, Dept. of Pharmacology and Therapeutics, McGill University, 3655 Drummond Street, Montreal, Quebec, Canada H3G 1Y6.
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ing development and in rapidly dividing cells. Glutathione is synthesized in the cell in two consecutive enzymatic reactions, catalyzed by y-glutamylcysteine synthetase and glutathione synthetase, both requiring ATP. Buthionine sulfoximine is a potent and specific inhibitor of the first enzyme, y-glutamylcysteine synthetase (Griffith and Meister, '79a; Griffith, '82). The administration of buthionine sulfoximine to mice or rats in vivo results in a dose- and time-dependent depletion of tissue glutathione levels (Griffith and Meister, '79b). Adult mice and rats can be given buthionine sulfoximine for several days with minimal signs of toxicity (Griffith, '82). Marked glutathione depletion with high doses of buthionine sulfoximine in adult mice and rats for periods of from 9 to 21 days is required before signs of overt toxicity such as degeneration in skeletal muscle, lung, and the epithelial cells on the intestine are seen (MBrtensson and Meister, '89; Mb-tensson et al., '90). Mitochondrial damage is found in the tissues that are affected. Perinatal animals appear to be more sensitive to depletion of glutathione with buthionine sulfoximine than adults. The administration of buthionine sulfoximine to male suckling mice caused lethargy, emaciation, and fur abnormalities (Calvin et al., '86). Treatment with buthionine sulfoximine (4mmolikg) on days 9 through 12 of life induced cataracts, while treatment on days 14 through 17 resulted in death, hind limb paralysis, and impaired spermatogenesis, but no cataracts. Treatment of rat pups on days 2 and 3 of life with buthionine sulfoximine (3 mmol/kg) also resulted in lens cataracts, while exposure on the first day of life was lethal to 50% of the pups (MBrtensson et al., '89). There is increasing evidence from in vitro studies that glutathione is important in modulating the response of the embryo to a variety of insults. Depletion of glutathione with buthionine sulfoximine in vitro in whole rat embryos cultured on day 10 of gestation for up to 45 h r potentiated the embryotoxicity of some teratogens such as acrolein (Slott and Hales, '87a), acetaminophen (Stark et al., '89), and cytochalasin D (Harris e t al., '88), but not that of others such as phosphoramide mustard (Slott and Hales, '87a), valproic acid (Harris et al., '88),7-hydroxy-2-acetylaminofluorene(Har-
ris e t al., '88), N-acetoxy-2-acetylaminofluorene (Faustman-Watts et al., '86; Stark et al., '89), or niridazole (Fantel et al., '89). Interestingly, depletion of glutathione itself with buthionine sulfoximine in this rat embryo culture system resulted in a n increase in malformations, but not embryolethality (Slott and Hales, '87b). Disturbance of the y-glutamyl cycle, and thus glutathione transport, synthesis, and degradation, by inhibition of y-glutamyltranspeptidase (with acivicin or goat anti-y-glutamyltranspeptidase antiserum) also resulted in embryotoxicity and malformations in cultured rat embryos (Stark e t al., '87). Thus, disturbances in glutathione homeostasis in vitro may have serious consequences with respect to embryonic development. The purpose of this study was to further investigate in vivo the effect of glutathione depletion with buthionine sulfoximine in pregnant rats. Buthionine sulfoximine was administered to timed pregnant rats on days 10 and 11of gestation, i.e., during the same phase of organogenesis a s the whole embryo culture system in vitro. Pregnancy outcome was assessed 1 day later, on day 12 of gestation, the stage of development equivalent to the end of the culture period in the previous in vitro experiments, as well as on day 20 of gestation. MATERIALS AND METHODS
Animals Timed-gestation pregnant Sprague-Dawley rats (200-225 g) were purchased from CharlesRiverCanadaInc. (St.Constant, Quebec). The day on which spermatozoa were found in the vaginal smear was considered day zero of pregnancy. Rats were housed in the McIntyre Animal Centre (McGill University, Montreal, Quebec) and given Purina r a t chow and water ad libidum. The rats were divided into three experimental groups. The control group received one dose of saline by gavage on each of days 10 and 11 of gestation. The buthionine sulfoximine treatment groups received a dose by gavage of either 4 or 8 mmol/kg body weight of buthionine sulfoximine in saline at 10 AM on both days 10 and 11of gestation. The L-buthionine-(S,R)-sulfoximine used in this experiment was purchased from Chemical Dynamics Corporation (Plainfield, New Jersey). Rats from each treatment group were killed at 2 PM on day 12 of gestation for the assessment of glutathione concentra-
GLUTATHIONE DEPLETION AND DEVELOPMENT
253
TABLE 1 . Glutathione concentrations in embryos and maternal tissues on day 12 ofgestation after treatment with buthionme sulfoximine on days 10 and 11 Glutathione concentration' Buthionine sulfoximine 4 mmolikg 8 mmolikg Tissue Control Embryo 90.7 i 23.4' 54.2 i 12.1 34.7 i 8.03 225.3 t 23.6 69.1 i 7.7" 47.1 2 8.P Muscle 58.5 t 17B3 44.9 t 8.73 Ovary 129.5 11.5 Liver 659.4 ? 156.9 518.2 t 64.0 342.0 2 62.5 Kidney 63.3 i 13.1 62.3 t 27.5 72.6 ? 38.1
*
'Glutathione concentrations are expressed as nmol glutathione equivalentsimg protein. 'Values are means k standard error of the mean (n = 5-6). 'Significantly ( P s 0 . 0 5 ) different from control by ANOVA with the F-test
tions and of pregnancy outcome (control, n = 6; buthionine sulfoximine, 4 mmol/kg, n = 6; 8 mmol/kg, n = 5). The remainder of the rats were killed on day 20 of gestation for the assessment of pregnancy outcome (control, n = 7; buthionine sulfoximine, 4 mmol/kg, n = 10; 8 mmollkg, n = 8).
Glutathione assays On day 12 of gestation, at 2 PM, pregnant rats from each treatment group were killed. Maternal ovaries and samples of maternal liver, kidney, and gastrocnemius muscle were frozen at -80°C for subsequent assay of glutathione and protein content. The uteri were removed and embryos were dissected free of decidua and their yolk sacs, examined for malformations and scored as described below, and then frozen until assayed individually for their glutathione content. (Dead or resorbed embryos were not assayed for glutathione.) Tissue aliquots were homogenized in 1 ml of 0.01 N HC1, and the total glutathione content (reduced and oxidized) of the homogenized samples was measured by the spectrophotometric method of Tietze ('69), as modified by Brehe and Burch ('76). Protein content was measured by the method of Lowry et al. ('51), with bovine serum albumin as the standard. Values were expressed as nmoles glutathione equivalentdmg protein. The level of detection of this assay was 0.016 nmol glutathione/mg protein. Assessment of pregnancy outcome To assess pregnancy outcome on day 12 of gestation pregnant rats from each of the three treatment groups ( n= 5-6lgroup) were killed and the uteri removed. The embryos were dissected free of decidua and the Reichert's membrane, leaving the yolk sac intact. The yolk sac circulation was exam-
ined and the diameter measured. Embryos were classified as either dead (no heart beat or yolk sac circulation) or alive, and living embryos a s normal or abnormal. A total morphological score was obtained for each embryo according to the morphological scoring system of Brown and Fabro ('81).Embryo size and stage were also assessed by measuring the crown rump length, the head length, and counting the number of somites. On day 20 of gestation the remaining rats in each treatment group ( n= 7-lOigroup) were killed. Implantation sites and the numbers of resorptions were counted. Living fetuses were removed, examined for external malformations and weighed.
Statistics Data were analyzed by one-way analysis of variance with the F-test to isolate the differences between groups (Glantz, '81). The unit for these analyses was the pregnant female or litter. The incidences of deaths and malformations per group were also analyzed with Fischer's exact test. The level of significance was P ~ 0 . 0 5 . RESULTS
Glutathione depletion in vivo by buthionine sulfoximine The glutathione concentrations in the embryos and in maternal tissues on day 12 of gestation, after treatment with saline or buthionine sulfoximine 52 and 28 h r earlier, are shown in Table 1. The low dose of buthionine sulfoximine produced a 40% decrease in embryonic glutathione concentrations while the high dose resulted in a 62% decrease. The effect on maternal tissues of exposure to buthionine sulfoximine on days 10 and 11of gestation was tissue dependent. Both doses of buthionine sulfoximine depressed glutathione concentrations in mus-
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TABLE 2. Pregnancy outcome as assessed on day 12 of gestation after treatment with buthionine sulfoximine on davs 10 and 11 Buthionine sulfoximine Control 4 mmolikg 8 mmolikg Implantationsipregnant female 13.0 5 1.3' 11.7 ? 0.8 10.6 ? 1.6 Live embryosipregnant female 12.5 t 1.2 11.0i 0.7 9.2 t 2.2 Dead or resorbed embryosipregnant female 0.5 i 0.2 0.8 ? 0.3 1.2 i 0.7 (Percent dead of total implanted embryos) (5.7%) (13.2%**) (3.9%) Malformed embryosipregnant female 0 0.3 i 0.2 2.0 i 1.3* (Percent malformed of total live embryos) (0%) (3.0%) (21.7%**) ~~
'Numbers are expressed as the means 5 standard error of the mean ( n = 5-6). *Significantly (P50.05) different from control by ANOVA with the F-test. **Significantly (P50.05)different from control by Fischer's exact test.
cle (4 mmol/kg, 69%; 8 mmol/kg, 79%) and in ovary (4mmol/kg, 55%;8 mmol/kg, 65%) to a similar extent. The glutathione content of maternal liver was depressed only 21% by the low dose of buthionine sulfoximine and 48% by the high dose; these values were not statistically significantly different from control. The glutathione content of the maternal kidney was not affected a t this time after treatment with buthionine sulfoximine (4mmol/kg, 102%; 8 mmol/kg, 115%).
Consequences on day 12 of gestation of buthionine sulfoximine The effects on day 12 of gestation of treatment with buthionine sulfoximine on days 10 and 11 are presented in Table 2. There was no significant effect of buthionine sulfoximine treatment on the number of implantations per pregnant female or on the number of living embryos per pregnant female. The number of dead or resorbed embryos was higher than control in the 8 mmol/kg buthionine sulfoximine treated group; this increase was significant by Fischer exact test. There was a dramatic increase in the number of malformed embryos per pregnant female in the group exposed to the high dose of buthionine sulfoximine. A total of 21.7% of the live embryos were malformed; malformed embryos were found in three out of five litters in this treatment group. Examples of some of the more severely malformed embryos and a control day 12 embryo are shown in Figure 1. The malformations observed were mainly in the brain region and consisted of shrunken or swollen hind brain, reduced prosencephalon, and blebs or blisters. The total morphological scores, according to the score system of Brown and Fabro ('81), of the control and buthionine sulfoxi-
mine exposed embryos are presented in Figure 2. Exposure to the buthionine sulfoximine on days 10 and 11 of gestation caused a dose-dependent decrease in the total morphological score of the embryos on day 12; this decrease was statistically significant in the higher dose group. A delay was observed in the development of a number of the parameters which make up the morphological score. This decrease was statistically significant only for the yolk sac score (control, 0.15, mean standard error of the 3.79 mean; buthionine sulfoximine, 4 mmol/kg, 2.78 2 0.26; 8 mmol/kg, 2.76 2 0.11; both buthionine sulfoximine-treated groups were significantly different from control) and otic system scores (control, 4.04 2 0.11; buthionine sulfoximine, 4 mmol/kg, 3.89 0.08; 8 mmollkg, 3.42 2 0.22; the buthionine sulfoximine treated high dose group was significantly different from control). Several parameters of the growth and developmental stage of the embryos on day 12 of gestation are presented in Table 3. There was no significant difference between the control and surviving buthionine sulfoximine exposed embryos with respect to yolk sac diameter, crown-rump or head length, or number of somite pairs.
*
*
*
Consequences on day 20 of gestation of buthionine sulfoximine The effects of exposure to buthionine sulfoximine on pregnancy outcome as assessed on day 20 of gestation are presented in Table 4. The numbers of implantations, resorptions, pups, and malformations per litter did not differ significantly from control in the buthionine sulfoximine exposed groups. There was also no effect of treatment with buthionine sulfoximine on the mean pup weight per litter on day 20 of gestation.
GLUTATHIONE DEPLETION AND DEVELOPMENT
255
Fig. 1. Effects of in viva glutathione depletion with buthionine sulfoximine on the day 12 rat embryo. Embryo A is a control embryo. Embryos B and C were exposed in utero to one dose of 8 mmolesikg of buthionine sulfoximine on each of days 10 and 11 of gestation.
DISCUSSION
Glutathione was still decreased in the embryo as well as in maternal muscle and ovary on day 12 of gestation, 28 h r after the second and last dose of buthionine sulfoximine, while there was no significant effect on glutathione concentrations in maternal liver or kidney. The rate of glutathione disappearance after buthionine sulfoximine treatment reflects the rate of glutathione turnover (Griffith and Meister, '79b). Glutathione turnover is highest in kidney, followed by liver; thus, i t is not surprising t h a t the glutathione concentration in kidney was completely back to control and that the concentration in liver, although depressed, was not significantly different from control by
28 h r after treatment with the second dose of buthionine sulfoximine. The rate of glutathione turnover in mouse skeletal muscle is also lower than t h a t of kidney (Griffith and Meister, '79b). The depletion of glutathione which was observed in this study in the embryos, maternal muscle, and ovary was in the range of 60%of control on day 12 of gestation. This observation would imply that the turnover of glutathione in these tissues is relatively slow. Previous investigations have found that there is no cell damage when glutathione is depleted even to 10% of control; mitochondria1 damage appeared when intracellular glutathione concentrations reached about 3% of control (MGrtensson and Meister, '89). The maxi-
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B.F. HALES AND H. BROWN
0
4
8
BSO Dose (rnrnoles/kg)
Fig. 2. The total morphological score, according to the score system of Brown and Fabro (’81),for rat embryos on day 12 of gestation. The mothers were treated with saline (control) or with buthionine sulfoximine by gavage on days 10 and 11 of gestation and killed on day 12. The values represent the means per litter -t the standard errors of the means ( n = 5-6igroup).
ma1 depletion of glutathione in the embryo in this experiment was not determined. The consequence to rat embryos in utero of a transitory depletion of glutathione during organogenesis was dependent on the de-
velopmental stage a t which they were examined. While a n increased incidence of deaths and malformations and a developmental delay was observed on day 12 of gestation, no effect of exposure to buthionine sulfoximine was still apparent on day 20 of gestation. This result is consistent with the results of a previous experiment, using mice done by Meister and his co-workers (MArtensson et al., ’89). These investigators found that when pregnant mice were treated with buthionine sulfoximine a t different periods during gestation (from days 6-10, 9-13, or 15-19) there were no malformations apparent on day 18 of gestation. The embryos in this study by MBrtensson et al. (’89) were not examined earlier than day 18 during gestation, so no “transitory malformations” would have been observed. Little is known about repair mechanisms in the embryo, and about the reversibility of any insult received during organogenesis. Glutathione deficiency does lead to suffcient cellular damage in sensitive organs in the embryo to cause deaths and malformations and a n apparent developmental delay on day 12 of gestation. In adult mice or rats treated with buthionine sulfoximine for periods from l to 2 weeks, the cellular damage observed when glutathione was depleted to less than 5% of control was usually mainly mitochondrial; these changes were revers-
TABLE 3 . Embryo growth parameters as assessed on day 12 ofgestation after treatment with buthionine sulfoximine on davs 10 and 11 Buthionine sulfoximine Control 4 mmolikp. 8 mmolike 6.7 ? 0.4’ 6.8 -t 0.4 6.0 ? 0.4 Yolk sac diameter (mm) Crown-rump length (mm) 5.9 L 0.2 6.0 -t 0.2 5.3 2 0.4 Head length (mm) 2.8 L 0.1 2.9 -t 0.2 2.6 t 0.1 46.3 2 0.9 46.8 ? 0.8 45.0 t 1.2 Number of somite pairs ‘Numbers are expressed as the means
-t
standard error of the mean ( n = 5-6)
TABLE 4. Pregnancy outcome as assessed on day 20 of gestation after treatment with buthionine sulfoximine on days 10 and 11 Buthionine sulfoximine Control 4 mmol/kg 8 mmoiikg 11.6 2 1.0’ 10.5 ? 1.1 11.1 2 0.5 Implantationsipregnant female 0.7 -t 0.3 0.9 ? 0.4 Resorptionsipregnant female 0.7 t 0.4 9.8 -t 1.2 10.3 ? 0.7 Pupdpregnant female 10.9 -t 1.3 0.1 -t 0.1 0.2 -t 0.2 0.1 -t 0.1 Malformationsipregnant female (Percent malformed of total live (1.3%) (2.0%) (1.2%) embryos) Mean pup weightilitter (8) 3.66 -t 0.20 3.54 +- 0.08 3.54 -t 0.08 ‘Numbers are expressed as the means
?
standard error of the mean (n = 7-10)
GLUTATHIONE DEPLETION AND DEVELOPMENT
ible and the administration of glutathione or of its monoester provided protection (MBrtensson et al., '90). During the time when glutathione was depleted in the rat embryos in this study, the embryos were undergoing a shift from mainly anaerobic glycolysis to the aerobic Krebs cycle electron transport system for energy metabolism (Shepard et al., '70). During this period of development the mitochondria have a rounded shape with few or no cristae, and change, with increased exposure to oxygen, to a n ovoid shape with a large number of parallel cristae (Morriss and New, '79). Thus, maturational changes in energy metabolism in embryos a t this stage of development may lead to a particularly high susceptibility to damage a s a consequence of glutathione depletion. The embryo undergoing organogenesis may be challenged with various endogenous reactive and toxic compounds to which it cannot respond when glutathione homeostasis is disturbed. ACKNOWLEDGMENTS
This work was supported by the Medical Research Council of Canada. LITERATURE CITED Brehe, J.E., and H.B. Burch (1976) Enzymatic assay for glutathione. Anal. Biochem., 74r189-197. Brown, N.A., and S. Fabro (1981) Quantitation of rat embryonic-development in vitro-a morphological scoring system. Teratology, 24:65-78. Calvin, H.I., C. Medvedovsky, and B.V. Worgul (1986) Near-total glutathione depletion and age-specific cataracts induced by buthionine sulfoximine in mice. Science, 233:553-555. Fantel, A.G., M.R. Juchau, J.W. Tracy, C.J. Burroughs, and R.E. Person (1989) Studies of mechanisms of niridazole-elicited embryotoxicity: evidence against a major role for covalent binding. Teratology, 39t63-74. Faustman-Watts, E.M., M.J. Namkung, and M.R. Juchau (1986) Modulation of the embryotoxicity in vitro of reactive metabolites of 2-acetylaminofluorene by reduced glutathione and ascorbate and via sulfation. Toxicol. Appl. Pharmacol., 86t400-410. Glantz, S.A. (1981) Primer of Biostatistics. McGrawHill, New York, p. 30. Griffth, O.W. (1982) Mechanism of action, metabolism and toxicity of buthionine sulfoximine and its higher homologs, potent inhibitors of glutathione synthesis. J. Biol. Chem., 257:13704-13712. Griffith, O.W., and A. Meister (1979a) Potent and specific inhibition of glutathione synthesis by buthionine sulfoximine (S-n-butyl homocysteine sulfoximine). J. Biol. Chem., 254:7558-7560. Griffith, O.W., and Meister, A. (1979133 Glutathione: Interorgan translocation, turnover and metabolism. Proc. Natl. Acad. Sci. U.S.A., 76t5606-5610. Harris, C., K.L. Stark, and M.R. Juchau (1988) Glutathione status and the incidence of neural tube de-
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fects elicited by direct acting teratogens in vitro. Teratology, 37577-590. Ishikawa, M., K. Sasaki, and Y. Takayanagi (1989) Injurious effect of buthionine sulfoximine, an inhibitor of glutathione biosynthesis, on the lethality and urotoxicity of cyclophosphamide in mice. Jpn. J. Pharmacol., 51:146-149. Ishikawa, M., G. Takayanagi, and K. Sasaki (1988) Effect of buthionine sulfoximine, an inhibitor of glutathione biosynthesis, on the selenium-induced lethality in mice. Jpn. J . Pharmacol., 48t283-286. Kuzuya, M., M. Naito, C. Funaki, T. Hayashi, K. Asai, and F. Kuzuya (1989) Protective role of intracellular glutathione against oxidized low density lipoprotein in cultured endothelial cells. Biochem. Biophys. Res. Commun., 163t1466-1472. Lowry, O.H., N.J. Rosebrough, A.L. Farr, and R.J. Randall (1951) Protein measurement with the Folin phenol reagent. J . Biol. Chem., 193265-275. Mlrtensson, J., A. Jain, and A. Meister (1990) Glutathione is required for intestinal function. Proc. Natl. Acad. Sci. U.S.A., 87:1715-1719. Mlrtensson, J., and A. Meister (1989) Mitochondria1 damage in muscle occurs after marked depletion of glutathione and is prevented by giving glutathione monoester. Proc. Natl. Acad. Sci. U.S.A., 86:471-475. MBrtensson, J., R. Steinherz, A. Jain, and A. Meister (1989) Glutathione ester prevents buthionine sulfoximine-induced cataracts and lens epithelial cell damage. Proc. Natl. Acad. Sci. U.S.A., 86t8727-8731. Mitchell, J.R., J.A. Cook, W. DeGraff, E. Glatstein, and A. Russo (1989) Keynote address: Glutathione modulation in cancer treatment: will it work? Int. J. Radiat. Oncol. Biol. Phys., 16t1289-1295. Moore, W.R., M.E. Anderson, A. Meister, K. Murata, and A. Kimura (1989) Increased capacity for glutathione synthesis enhances resistance to radiation in Escherichia coli: A possible model for mammalian cell protection. Proc. Natl. Acad. Sci. U.S.A., 86: 1461-1464. Morriss, G.M., and D.A.T. New (1979) Effect of oxygen concentration on morphogenesis of cranial neural folds and neural crest in cultured rat embryos. J . Embryol. Exp. Morphol., 54:17-35. Shepard, T.H., T. Tanimura, and M.A. Robkin (1970) Energy metabolism in early mammalian embryos. Dev. Biol. Suppl., 4t42-58. Singhal, R.K., M.E. Anderson, and A. Meister (1987) Glutathione, a first line of defense against cadmium toxicity. FASEB J., 1t220-223. Slott, V.L., and B.F. Hales (1987a) Enhancement of the embryotoxicity of acrolein, but not phosphoramide mustard, by glutathione depletion in rat embryos in vitro. Biochem. Pharmacol., 36t2019-2025. Slott, V.L., and B.F. Hales (1987b) Effect of glutathione depletion by buthionine sulfoximine on rat embryonic development in vitro. Biochem. Pharmacol., 36t683688. Stark, K.L., C. Harris, and M.R. Juchau (1987) Embryotoxicity elicited by inhibition of y-glutamyltransferase by acivicin and transferase antibodies in cultured rat embryos. Toxicol. Appl. Pharmacol., 89:88-96. Stark, K.L., C. Harris, and M.R. Juchau (1989) Influence of electrophilic character and glutathione depletion on chemical dysmorphogenesis in cultured rat embryos. Biochem. Pharmacol., 38:2685-2692. Tietze, F. (1969) Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione. Applications to mammalian blood and other tissues. Anal. Biochem., 27:502-522.
The Effect of In Vivo Glutathione Depletion With Buthionine Sulfoximine on Rat Embryo Development BARBARA F. HALES AND HEATHER BROWN Department of Pharmacology and Therapeutics, McGill University, 3655 Drummond Street, Montrt!al, Quebec, Canada H3G 1 Y6
ABSTRACT
Glutathione is a n abundant endogenous nucleophile whose depletion may have adverse effects on a number of cellular processes, including protein and DNA synthesis, amino acid transport, and detoxification of reactive electrophiles. Previous studies have indicated that a certain basal level of glutathione is essential for normal development in vitro in the whole rat embryo culture system. The objective of this study was to determine the effect of glutathione depletion with buthionine sulfoximine on the development of rat embryos in vivo. Timed pregnant Sprague-Dawley rats were treated by gavage on days 10 and 11 of gestation with saline (control) or with L-buthionine-(S,R)sulfoximine (4or 8 mmol/kg). Pregnancy outcome was assessed on day 12 or day 20 of gestation. The glutathione content of embryos and of maternal organs was measured on day 12. Glutathione concentrations were significantly decreased in the embryos and in maternal muscle and ovary but not in maternal liver and kidney. Glutathione depletion, when assessed on day 12 of gestation, was found to result in a n increase in the numbers of dead and malformed embryos. Treatment with the higher dose of buthionine sulfoximine resulted in the death of 13.2% of the total implanted embryos; of the surviving embryos, 21.7% were malformed. There was also a significant decrease on day 12 after treatment with the higher dose of buthionine sulfoximine in the embryonic total morphological score, a measure of the developmental stage of the embryos (control, 57.721.0; 8 mmolikg buthionine sulfoximine, 52.82 1.8).No significant effect of treatment with either dose of buthionine sulfoximine with respect to growth parameters was observed on day 12 of gestation. Interestingly, no significant effect of treatment with either dose of buthionine sulfoximine on numbers of pups, resorptions, malformations, or fetal weight was found in the litters examined on day 20 of gestation. Thus, after glutathione depletion with buthionine sulfoximine, a n increase in malformations and developmental delay is found on day 12 of gestation; however the malformations observed in day 12 embryos apparently do not persist during later development.
Glutathione is a tripeptide abundant in the cells of most organisms. This nucleophile is thought to be a n essential cell component, playing a n important role as a cellular antioxidant as well a s in amino acid transport. Increased intracellular glutathione protects the cell against the toxic effects of a variety of drugs and of radiation. In a recent report, a n increased capacity to synthesize glutathione in a model strain of E . coli was associated with enhanced resistance to y-irradiation (Moore et al., '89). Decreased glutathione synthesis or content re0 1991 WILEY-LISS,
INC.
sults in greater sensitivity to heavy metals (Singhal et al., '87; Ishikawa et al., '881, endogenous electrophiles (Kuzuya et al., '89), cancer chemotherapeutic agents, and radiation (Ishikawa et al., '89; Mitchell et al., '89). The maintenance of reduced glutathione may be particularly important dur-
Received October 10, 1990; accepted March 19. 1991. Address reprint requests to Dr. B. F. Hales, Dept. of Pharmacology and Therapeutics, McGill University, 3655 Drummond Street, Montreal, Quebec, Canada H3G 1Y6.
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ing development and in rapidly dividing cells. Glutathione is synthesized in the cell in two consecutive enzymatic reactions, catalyzed by y-glutamylcysteine synthetase and glutathione synthetase, both requiring ATP. Buthionine sulfoximine is a potent and specific inhibitor of the first enzyme, y-glutamylcysteine synthetase (Griffith and Meister, '79a; Griffith, '82). The administration of buthionine sulfoximine to mice or rats in vivo results in a dose- and time-dependent depletion of tissue glutathione levels (Griffith and Meister, '79b). Adult mice and rats can be given buthionine sulfoximine for several days with minimal signs of toxicity (Griffith, '82). Marked glutathione depletion with high doses of buthionine sulfoximine in adult mice and rats for periods of from 9 to 21 days is required before signs of overt toxicity such as degeneration in skeletal muscle, lung, and the epithelial cells on the intestine are seen (MBrtensson and Meister, '89; Mb-tensson et al., '90). Mitochondrial damage is found in the tissues that are affected. Perinatal animals appear to be more sensitive to depletion of glutathione with buthionine sulfoximine than adults. The administration of buthionine sulfoximine to male suckling mice caused lethargy, emaciation, and fur abnormalities (Calvin et al., '86). Treatment with buthionine sulfoximine (4mmolikg) on days 9 through 12 of life induced cataracts, while treatment on days 14 through 17 resulted in death, hind limb paralysis, and impaired spermatogenesis, but no cataracts. Treatment of rat pups on days 2 and 3 of life with buthionine sulfoximine (3 mmol/kg) also resulted in lens cataracts, while exposure on the first day of life was lethal to 50% of the pups (MBrtensson et al., '89). There is increasing evidence from in vitro studies that glutathione is important in modulating the response of the embryo to a variety of insults. Depletion of glutathione with buthionine sulfoximine in vitro in whole rat embryos cultured on day 10 of gestation for up to 45 h r potentiated the embryotoxicity of some teratogens such as acrolein (Slott and Hales, '87a), acetaminophen (Stark et al., '89), and cytochalasin D (Harris e t al., '88), but not that of others such as phosphoramide mustard (Slott and Hales, '87a), valproic acid (Harris et al., '88),7-hydroxy-2-acetylaminofluorene(Har-
ris e t al., '88), N-acetoxy-2-acetylaminofluorene (Faustman-Watts et al., '86; Stark et al., '89), or niridazole (Fantel et al., '89). Interestingly, depletion of glutathione itself with buthionine sulfoximine in this rat embryo culture system resulted in a n increase in malformations, but not embryolethality (Slott and Hales, '87b). Disturbance of the y-glutamyl cycle, and thus glutathione transport, synthesis, and degradation, by inhibition of y-glutamyltranspeptidase (with acivicin or goat anti-y-glutamyltranspeptidase antiserum) also resulted in embryotoxicity and malformations in cultured rat embryos (Stark e t al., '87). Thus, disturbances in glutathione homeostasis in vitro may have serious consequences with respect to embryonic development. The purpose of this study was to further investigate in vivo the effect of glutathione depletion with buthionine sulfoximine in pregnant rats. Buthionine sulfoximine was administered to timed pregnant rats on days 10 and 11of gestation, i.e., during the same phase of organogenesis a s the whole embryo culture system in vitro. Pregnancy outcome was assessed 1 day later, on day 12 of gestation, the stage of development equivalent to the end of the culture period in the previous in vitro experiments, as well as on day 20 of gestation. MATERIALS AND METHODS
Animals Timed-gestation pregnant Sprague-Dawley rats (200-225 g) were purchased from CharlesRiverCanadaInc. (St.Constant, Quebec). The day on which spermatozoa were found in the vaginal smear was considered day zero of pregnancy. Rats were housed in the McIntyre Animal Centre (McGill University, Montreal, Quebec) and given Purina r a t chow and water ad libidum. The rats were divided into three experimental groups. The control group received one dose of saline by gavage on each of days 10 and 11 of gestation. The buthionine sulfoximine treatment groups received a dose by gavage of either 4 or 8 mmol/kg body weight of buthionine sulfoximine in saline at 10 AM on both days 10 and 11of gestation. The L-buthionine-(S,R)-sulfoximine used in this experiment was purchased from Chemical Dynamics Corporation (Plainfield, New Jersey). Rats from each treatment group were killed at 2 PM on day 12 of gestation for the assessment of glutathione concentra-
GLUTATHIONE DEPLETION AND DEVELOPMENT
253
TABLE 1 . Glutathione concentrations in embryos and maternal tissues on day 12 ofgestation after treatment with buthionme sulfoximine on days 10 and 11 Glutathione concentration' Buthionine sulfoximine 4 mmolikg 8 mmolikg Tissue Control Embryo 90.7 i 23.4' 54.2 i 12.1 34.7 i 8.03 225.3 t 23.6 69.1 i 7.7" 47.1 2 8.P Muscle 58.5 t 17B3 44.9 t 8.73 Ovary 129.5 11.5 Liver 659.4 ? 156.9 518.2 t 64.0 342.0 2 62.5 Kidney 63.3 i 13.1 62.3 t 27.5 72.6 ? 38.1
*
'Glutathione concentrations are expressed as nmol glutathione equivalentsimg protein. 'Values are means k standard error of the mean (n = 5-6). 'Significantly ( P s 0 . 0 5 ) different from control by ANOVA with the F-test
tions and of pregnancy outcome (control, n = 6; buthionine sulfoximine, 4 mmol/kg, n = 6; 8 mmol/kg, n = 5). The remainder of the rats were killed on day 20 of gestation for the assessment of pregnancy outcome (control, n = 7; buthionine sulfoximine, 4 mmol/kg, n = 10; 8 mmollkg, n = 8).
Glutathione assays On day 12 of gestation, at 2 PM, pregnant rats from each treatment group were killed. Maternal ovaries and samples of maternal liver, kidney, and gastrocnemius muscle were frozen at -80°C for subsequent assay of glutathione and protein content. The uteri were removed and embryos were dissected free of decidua and their yolk sacs, examined for malformations and scored as described below, and then frozen until assayed individually for their glutathione content. (Dead or resorbed embryos were not assayed for glutathione.) Tissue aliquots were homogenized in 1 ml of 0.01 N HC1, and the total glutathione content (reduced and oxidized) of the homogenized samples was measured by the spectrophotometric method of Tietze ('69), as modified by Brehe and Burch ('76). Protein content was measured by the method of Lowry et al. ('51), with bovine serum albumin as the standard. Values were expressed as nmoles glutathione equivalentdmg protein. The level of detection of this assay was 0.016 nmol glutathione/mg protein. Assessment of pregnancy outcome To assess pregnancy outcome on day 12 of gestation pregnant rats from each of the three treatment groups ( n= 5-6lgroup) were killed and the uteri removed. The embryos were dissected free of decidua and the Reichert's membrane, leaving the yolk sac intact. The yolk sac circulation was exam-
ined and the diameter measured. Embryos were classified as either dead (no heart beat or yolk sac circulation) or alive, and living embryos a s normal or abnormal. A total morphological score was obtained for each embryo according to the morphological scoring system of Brown and Fabro ('81).Embryo size and stage were also assessed by measuring the crown rump length, the head length, and counting the number of somites. On day 20 of gestation the remaining rats in each treatment group ( n= 7-lOigroup) were killed. Implantation sites and the numbers of resorptions were counted. Living fetuses were removed, examined for external malformations and weighed.
Statistics Data were analyzed by one-way analysis of variance with the F-test to isolate the differences between groups (Glantz, '81). The unit for these analyses was the pregnant female or litter. The incidences of deaths and malformations per group were also analyzed with Fischer's exact test. The level of significance was P ~ 0 . 0 5 . RESULTS
Glutathione depletion in vivo by buthionine sulfoximine The glutathione concentrations in the embryos and in maternal tissues on day 12 of gestation, after treatment with saline or buthionine sulfoximine 52 and 28 h r earlier, are shown in Table 1. The low dose of buthionine sulfoximine produced a 40% decrease in embryonic glutathione concentrations while the high dose resulted in a 62% decrease. The effect on maternal tissues of exposure to buthionine sulfoximine on days 10 and 11of gestation was tissue dependent. Both doses of buthionine sulfoximine depressed glutathione concentrations in mus-
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TABLE 2. Pregnancy outcome as assessed on day 12 of gestation after treatment with buthionine sulfoximine on davs 10 and 11 Buthionine sulfoximine Control 4 mmolikg 8 mmolikg Implantationsipregnant female 13.0 5 1.3' 11.7 ? 0.8 10.6 ? 1.6 Live embryosipregnant female 12.5 t 1.2 11.0i 0.7 9.2 t 2.2 Dead or resorbed embryosipregnant female 0.5 i 0.2 0.8 ? 0.3 1.2 i 0.7 (Percent dead of total implanted embryos) (5.7%) (13.2%**) (3.9%) Malformed embryosipregnant female 0 0.3 i 0.2 2.0 i 1.3* (Percent malformed of total live embryos) (0%) (3.0%) (21.7%**) ~~
'Numbers are expressed as the means 5 standard error of the mean ( n = 5-6). *Significantly (P50.05) different from control by ANOVA with the F-test. **Significantly (P50.05)different from control by Fischer's exact test.
cle (4 mmol/kg, 69%; 8 mmol/kg, 79%) and in ovary (4mmol/kg, 55%;8 mmol/kg, 65%) to a similar extent. The glutathione content of maternal liver was depressed only 21% by the low dose of buthionine sulfoximine and 48% by the high dose; these values were not statistically significantly different from control. The glutathione content of the maternal kidney was not affected a t this time after treatment with buthionine sulfoximine (4mmol/kg, 102%; 8 mmol/kg, 115%).
Consequences on day 12 of gestation of buthionine sulfoximine The effects on day 12 of gestation of treatment with buthionine sulfoximine on days 10 and 11 are presented in Table 2. There was no significant effect of buthionine sulfoximine treatment on the number of implantations per pregnant female or on the number of living embryos per pregnant female. The number of dead or resorbed embryos was higher than control in the 8 mmol/kg buthionine sulfoximine treated group; this increase was significant by Fischer exact test. There was a dramatic increase in the number of malformed embryos per pregnant female in the group exposed to the high dose of buthionine sulfoximine. A total of 21.7% of the live embryos were malformed; malformed embryos were found in three out of five litters in this treatment group. Examples of some of the more severely malformed embryos and a control day 12 embryo are shown in Figure 1. The malformations observed were mainly in the brain region and consisted of shrunken or swollen hind brain, reduced prosencephalon, and blebs or blisters. The total morphological scores, according to the score system of Brown and Fabro ('81), of the control and buthionine sulfoxi-
mine exposed embryos are presented in Figure 2. Exposure to the buthionine sulfoximine on days 10 and 11 of gestation caused a dose-dependent decrease in the total morphological score of the embryos on day 12; this decrease was statistically significant in the higher dose group. A delay was observed in the development of a number of the parameters which make up the morphological score. This decrease was statistically significant only for the yolk sac score (control, 0.15, mean standard error of the 3.79 mean; buthionine sulfoximine, 4 mmol/kg, 2.78 2 0.26; 8 mmol/kg, 2.76 2 0.11; both buthionine sulfoximine-treated groups were significantly different from control) and otic system scores (control, 4.04 2 0.11; buthionine sulfoximine, 4 mmol/kg, 3.89 0.08; 8 mmollkg, 3.42 2 0.22; the buthionine sulfoximine treated high dose group was significantly different from control). Several parameters of the growth and developmental stage of the embryos on day 12 of gestation are presented in Table 3. There was no significant difference between the control and surviving buthionine sulfoximine exposed embryos with respect to yolk sac diameter, crown-rump or head length, or number of somite pairs.
*
*
*
Consequences on day 20 of gestation of buthionine sulfoximine The effects of exposure to buthionine sulfoximine on pregnancy outcome as assessed on day 20 of gestation are presented in Table 4. The numbers of implantations, resorptions, pups, and malformations per litter did not differ significantly from control in the buthionine sulfoximine exposed groups. There was also no effect of treatment with buthionine sulfoximine on the mean pup weight per litter on day 20 of gestation.
GLUTATHIONE DEPLETION AND DEVELOPMENT
255
Fig. 1. Effects of in viva glutathione depletion with buthionine sulfoximine on the day 12 rat embryo. Embryo A is a control embryo. Embryos B and C were exposed in utero to one dose of 8 mmolesikg of buthionine sulfoximine on each of days 10 and 11 of gestation.
DISCUSSION
Glutathione was still decreased in the embryo as well as in maternal muscle and ovary on day 12 of gestation, 28 h r after the second and last dose of buthionine sulfoximine, while there was no significant effect on glutathione concentrations in maternal liver or kidney. The rate of glutathione disappearance after buthionine sulfoximine treatment reflects the rate of glutathione turnover (Griffith and Meister, '79b). Glutathione turnover is highest in kidney, followed by liver; thus, i t is not surprising t h a t the glutathione concentration in kidney was completely back to control and that the concentration in liver, although depressed, was not significantly different from control by
28 h r after treatment with the second dose of buthionine sulfoximine. The rate of glutathione turnover in mouse skeletal muscle is also lower than t h a t of kidney (Griffith and Meister, '79b). The depletion of glutathione which was observed in this study in the embryos, maternal muscle, and ovary was in the range of 60%of control on day 12 of gestation. This observation would imply that the turnover of glutathione in these tissues is relatively slow. Previous investigations have found that there is no cell damage when glutathione is depleted even to 10% of control; mitochondria1 damage appeared when intracellular glutathione concentrations reached about 3% of control (MGrtensson and Meister, '89). The maxi-
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B.F. HALES AND H. BROWN
0
4
8
BSO Dose (rnrnoles/kg)
Fig. 2. The total morphological score, according to the score system of Brown and Fabro (’81),for rat embryos on day 12 of gestation. The mothers were treated with saline (control) or with buthionine sulfoximine by gavage on days 10 and 11 of gestation and killed on day 12. The values represent the means per litter -t the standard errors of the means ( n = 5-6igroup).
ma1 depletion of glutathione in the embryo in this experiment was not determined. The consequence to rat embryos in utero of a transitory depletion of glutathione during organogenesis was dependent on the de-
velopmental stage a t which they were examined. While a n increased incidence of deaths and malformations and a developmental delay was observed on day 12 of gestation, no effect of exposure to buthionine sulfoximine was still apparent on day 20 of gestation. This result is consistent with the results of a previous experiment, using mice done by Meister and his co-workers (MArtensson et al., ’89). These investigators found that when pregnant mice were treated with buthionine sulfoximine a t different periods during gestation (from days 6-10, 9-13, or 15-19) there were no malformations apparent on day 18 of gestation. The embryos in this study by MBrtensson et al. (’89) were not examined earlier than day 18 during gestation, so no “transitory malformations” would have been observed. Little is known about repair mechanisms in the embryo, and about the reversibility of any insult received during organogenesis. Glutathione deficiency does lead to suffcient cellular damage in sensitive organs in the embryo to cause deaths and malformations and a n apparent developmental delay on day 12 of gestation. In adult mice or rats treated with buthionine sulfoximine for periods from l to 2 weeks, the cellular damage observed when glutathione was depleted to less than 5% of control was usually mainly mitochondrial; these changes were revers-
TABLE 3 . Embryo growth parameters as assessed on day 12 ofgestation after treatment with buthionine sulfoximine on davs 10 and 11 Buthionine sulfoximine Control 4 mmolikp. 8 mmolike 6.7 ? 0.4’ 6.8 -t 0.4 6.0 ? 0.4 Yolk sac diameter (mm) Crown-rump length (mm) 5.9 L 0.2 6.0 -t 0.2 5.3 2 0.4 Head length (mm) 2.8 L 0.1 2.9 -t 0.2 2.6 t 0.1 46.3 2 0.9 46.8 ? 0.8 45.0 t 1.2 Number of somite pairs ‘Numbers are expressed as the means
-t
standard error of the mean ( n = 5-6)
TABLE 4. Pregnancy outcome as assessed on day 20 of gestation after treatment with buthionine sulfoximine on days 10 and 11 Buthionine sulfoximine Control 4 mmol/kg 8 mmoiikg 11.6 2 1.0’ 10.5 ? 1.1 11.1 2 0.5 Implantationsipregnant female 0.7 -t 0.3 0.9 ? 0.4 Resorptionsipregnant female 0.7 t 0.4 9.8 -t 1.2 10.3 ? 0.7 Pupdpregnant female 10.9 -t 1.3 0.1 -t 0.1 0.2 -t 0.2 0.1 -t 0.1 Malformationsipregnant female (Percent malformed of total live (1.3%) (2.0%) (1.2%) embryos) Mean pup weightilitter (8) 3.66 -t 0.20 3.54 +- 0.08 3.54 -t 0.08 ‘Numbers are expressed as the means
?
standard error of the mean (n = 7-10)
GLUTATHIONE DEPLETION AND DEVELOPMENT
ible and the administration of glutathione or of its monoester provided protection (MBrtensson et al., '90). During the time when glutathione was depleted in the rat embryos in this study, the embryos were undergoing a shift from mainly anaerobic glycolysis to the aerobic Krebs cycle electron transport system for energy metabolism (Shepard et al., '70). During this period of development the mitochondria have a rounded shape with few or no cristae, and change, with increased exposure to oxygen, to a n ovoid shape with a large number of parallel cristae (Morriss and New, '79). Thus, maturational changes in energy metabolism in embryos a t this stage of development may lead to a particularly high susceptibility to damage a s a consequence of glutathione depletion. The embryo undergoing organogenesis may be challenged with various endogenous reactive and toxic compounds to which it cannot respond when glutathione homeostasis is disturbed. ACKNOWLEDGMENTS
This work was supported by the Medical Research Council of Canada. LITERATURE CITED Brehe, J.E., and H.B. Burch (1976) Enzymatic assay for glutathione. Anal. Biochem., 74r189-197. Brown, N.A., and S. Fabro (1981) Quantitation of rat embryonic-development in vitro-a morphological scoring system. Teratology, 24:65-78. Calvin, H.I., C. Medvedovsky, and B.V. Worgul (1986) Near-total glutathione depletion and age-specific cataracts induced by buthionine sulfoximine in mice. Science, 233:553-555. Fantel, A.G., M.R. Juchau, J.W. Tracy, C.J. Burroughs, and R.E. Person (1989) Studies of mechanisms of niridazole-elicited embryotoxicity: evidence against a major role for covalent binding. Teratology, 39t63-74. Faustman-Watts, E.M., M.J. Namkung, and M.R. Juchau (1986) Modulation of the embryotoxicity in vitro of reactive metabolites of 2-acetylaminofluorene by reduced glutathione and ascorbate and via sulfation. Toxicol. Appl. Pharmacol., 86t400-410. Glantz, S.A. (1981) Primer of Biostatistics. McGrawHill, New York, p. 30. Griffth, O.W. (1982) Mechanism of action, metabolism and toxicity of buthionine sulfoximine and its higher homologs, potent inhibitors of glutathione synthesis. J. Biol. Chem., 257:13704-13712. Griffith, O.W., and A. Meister (1979a) Potent and specific inhibition of glutathione synthesis by buthionine sulfoximine (S-n-butyl homocysteine sulfoximine). J. Biol. Chem., 254:7558-7560. Griffith, O.W., and Meister, A. (1979133 Glutathione: Interorgan translocation, turnover and metabolism. Proc. Natl. Acad. Sci. U.S.A., 76t5606-5610. Harris, C., K.L. Stark, and M.R. Juchau (1988) Glutathione status and the incidence of neural tube de-
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fects elicited by direct acting teratogens in vitro. Teratology, 37577-590. Ishikawa, M., K. Sasaki, and Y. Takayanagi (1989) Injurious effect of buthionine sulfoximine, an inhibitor of glutathione biosynthesis, on the lethality and urotoxicity of cyclophosphamide in mice. Jpn. J. Pharmacol., 51:146-149. Ishikawa, M., G. Takayanagi, and K. Sasaki (1988) Effect of buthionine sulfoximine, an inhibitor of glutathione biosynthesis, on the selenium-induced lethality in mice. Jpn. J . Pharmacol., 48t283-286. Kuzuya, M., M. Naito, C. Funaki, T. Hayashi, K. Asai, and F. Kuzuya (1989) Protective role of intracellular glutathione against oxidized low density lipoprotein in cultured endothelial cells. Biochem. Biophys. Res. Commun., 163t1466-1472. Lowry, O.H., N.J. Rosebrough, A.L. Farr, and R.J. Randall (1951) Protein measurement with the Folin phenol reagent. J . Biol. Chem., 193265-275. Mlrtensson, J., A. Jain, and A. Meister (1990) Glutathione is required for intestinal function. Proc. Natl. Acad. Sci. U.S.A., 87:1715-1719. Mlrtensson, J., and A. Meister (1989) Mitochondria1 damage in muscle occurs after marked depletion of glutathione and is prevented by giving glutathione monoester. Proc. Natl. Acad. Sci. U.S.A., 86:471-475. MBrtensson, J., R. Steinherz, A. Jain, and A. Meister (1989) Glutathione ester prevents buthionine sulfoximine-induced cataracts and lens epithelial cell damage. Proc. Natl. Acad. Sci. U.S.A., 86t8727-8731. Mitchell, J.R., J.A. Cook, W. DeGraff, E. Glatstein, and A. Russo (1989) Keynote address: Glutathione modulation in cancer treatment: will it work? Int. J. Radiat. Oncol. Biol. Phys., 16t1289-1295. Moore, W.R., M.E. Anderson, A. Meister, K. Murata, and A. Kimura (1989) Increased capacity for glutathione synthesis enhances resistance to radiation in Escherichia coli: A possible model for mammalian cell protection. Proc. Natl. Acad. Sci. U.S.A., 86: 1461-1464. Morriss, G.M., and D.A.T. New (1979) Effect of oxygen concentration on morphogenesis of cranial neural folds and neural crest in cultured rat embryos. J . Embryol. Exp. Morphol., 54:17-35. Shepard, T.H., T. Tanimura, and M.A. Robkin (1970) Energy metabolism in early mammalian embryos. Dev. Biol. Suppl., 4t42-58. Singhal, R.K., M.E. Anderson, and A. Meister (1987) Glutathione, a first line of defense against cadmium toxicity. FASEB J., 1t220-223. Slott, V.L., and B.F. Hales (1987a) Enhancement of the embryotoxicity of acrolein, but not phosphoramide mustard, by glutathione depletion in rat embryos in vitro. Biochem. Pharmacol., 36t2019-2025. Slott, V.L., and B.F. Hales (1987b) Effect of glutathione depletion by buthionine sulfoximine on rat embryonic development in vitro. Biochem. Pharmacol., 36t683688. Stark, K.L., C. Harris, and M.R. Juchau (1987) Embryotoxicity elicited by inhibition of y-glutamyltransferase by acivicin and transferase antibodies in cultured rat embryos. Toxicol. Appl. Pharmacol., 89:88-96. Stark, K.L., C. Harris, and M.R. Juchau (1989) Influence of electrophilic character and glutathione depletion on chemical dysmorphogenesis in cultured rat embryos. Biochem. Pharmacol., 38:2685-2692. Tietze, F. (1969) Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione. Applications to mammalian blood and other tissues. Anal. Biochem., 27:502-522.
