TERATOLOGY 41:275-280 (1990)
Effects of Phorone and/or Buthionine Sulfoximine on Teratogenicity of 5-Fluorouracil in Mice MASATO NAYA, YOUICHI MATAKI, HIROSHI TAKAHIRA, TAKASHI DEGUCHI, AND MINE0 YASUDA Kyowa Toxicological Research Laboratories, Yamaguchi 755, Japan (M.N., Y.M., H.T., T.D.);Department of Anatomy, Hiroshima Uniuersity School of Medicine, Hiroshima 734, Japan (M.N., M.Y.)
ABSTRACT Embryotoxicity and teratogenicity of 5-f luorouracil (5-FU) and modulation of its effect by the depletors of glutathione (GSH) were evaluated in mice. Pregnant ICR mice were intraperitoneally (i.p.) injected with 25 mgkg of 5-FU on day 11 of gestation (vaginal plug = day 0). Mice were pretreated i.p. with 250 mg/kg of phorone, a GSH depleting agent and/or 200 mgkg of buthionine sulfoximine (BSO, an inhibitor of GSH biosynthesis) 4 hours before dosing with 5-FU. Dams were killed on day 17 of gestation. Fetuses were examined for external malformations, especially limb malformations. Pretreatment with phorone or BSO decreased fetal weight and increased the frequency and severity of oligodactyly induced by 5-FU, as well as the reduction of maternal GSH levels. Combined use of 125 mgikg phorone and 100 mg/kg BSO i.p. augmented growth retardation induced with 5-FU. Cotreatment with exogenous GSH, at a dose of 300 mg/kg injected intravenously, could not suppress the augmentative effects of phorone and/or BSO on 5-FU teratogenicity under these experimental conditions. These results indicate that the level of endogenous GSH is one of the factors which significantly affects teratogenicity of 5-FU. Glutathione (GSH), y-glutamylcysteinylglycine, the most abundant nonprotein thiol in cells, is known to be a major intracellular antioxidant, a key component in the metabolism of cysteine and cysteine-containing proteins, and a specific deactivator of potentially toxic electrophilic agents. A number of compounds have been developed to alter the metabolism and intracellular concentration of GSH. The intracellular GSH levels in different cell systems can be decreased after treatment with diethylmaleate (DEM),phorone (diisopropylidene acetone), and D,Lbuthionine-S-R-sulfoximine(BSO) (Meister, '85). Teratogenicity of the antitumor agent 5fluorouracil (5-FU) has been characterized by inducing limb malformations in mice (Dagg, '60; Dagg et al., '66; Katagiri, '83). There is a considerable advantage in using limb malformations to evaluate the protective and/or augmentative effect of chemicals on teratogenicity, because severity of limb malformations can be easily defined by the number of missing digits. We have previ-
0 1990 WILEY-LISS, INC.
ously shown that GSH depletion with DEM increases the incidence of oligodactyly; on the contrary, pretreatment with GSH decrease the incidence of oligodactyly induced with 5-FU in mice (Naya et al., '87). DEM, however, inhibited microsomal p-450-dependent enzymes, as well as depleting GSH, thus interfering with the bioactivation of halogenated hydrocarbons (Anders, '78; Suarez et al., '81).Therefore, DEM may deplete endogenous GSH nonspecifically. On the other hand, phorone depletes GSH but does not interfere with the microsomal mixed-function oxidase system (Siegers et al., '85). Recently, BSO, the specific inhibitor of y-glutamyl cysteine synthetase, has been used extensively to deplete cellular GSH in studies designed to enhance the toxic effects of various antitumor agents (Arrick and Nathan, '84; Russo et al., '86). In the present study, we used phorone and BSO in order to investigate the relationship of GSH levels to 5-FU teratogenicity in mice. Received June 12, 1989; accepted September 18, 1989
276
M. NAYA ET AL 2o
r
T
L
Phorone
-
C P h o r o n e t BSO
** I
OL I
0
I 2
I 4
I 6
!c-------I 24
Hours a f t e r t r e a t m e n t Fig. 1. Effects of phorone and/or buthionine sulfoximine (BSO) on glutathione (GSH) levels in maternal serum of ICR mice. Phorone 250 mg/kg i.p., BSO 200 mgikg i.p., or phorone 125 mgikg plus BSO 100 mgikg i.p. was administered on day 11 of gestation. Pregnant mice were killed a t 0, 2, 4,6, or 24 hours after administration of phorone and/or BSO. Levels of GSH were determined by high performance liquid chromatography. Each value represents the mean i S.D. of four animals. *P
Effects of Phorone and/or Buthionine Sulfoximine on Teratogenicity of 5-Fluorouracil in Mice MASATO NAYA, YOUICHI MATAKI, HIROSHI TAKAHIRA, TAKASHI DEGUCHI, AND MINE0 YASUDA Kyowa Toxicological Research Laboratories, Yamaguchi 755, Japan (M.N., Y.M., H.T., T.D.);Department of Anatomy, Hiroshima Uniuersity School of Medicine, Hiroshima 734, Japan (M.N., M.Y.)
ABSTRACT Embryotoxicity and teratogenicity of 5-f luorouracil (5-FU) and modulation of its effect by the depletors of glutathione (GSH) were evaluated in mice. Pregnant ICR mice were intraperitoneally (i.p.) injected with 25 mgkg of 5-FU on day 11 of gestation (vaginal plug = day 0). Mice were pretreated i.p. with 250 mg/kg of phorone, a GSH depleting agent and/or 200 mgkg of buthionine sulfoximine (BSO, an inhibitor of GSH biosynthesis) 4 hours before dosing with 5-FU. Dams were killed on day 17 of gestation. Fetuses were examined for external malformations, especially limb malformations. Pretreatment with phorone or BSO decreased fetal weight and increased the frequency and severity of oligodactyly induced by 5-FU, as well as the reduction of maternal GSH levels. Combined use of 125 mgikg phorone and 100 mg/kg BSO i.p. augmented growth retardation induced with 5-FU. Cotreatment with exogenous GSH, at a dose of 300 mg/kg injected intravenously, could not suppress the augmentative effects of phorone and/or BSO on 5-FU teratogenicity under these experimental conditions. These results indicate that the level of endogenous GSH is one of the factors which significantly affects teratogenicity of 5-FU. Glutathione (GSH), y-glutamylcysteinylglycine, the most abundant nonprotein thiol in cells, is known to be a major intracellular antioxidant, a key component in the metabolism of cysteine and cysteine-containing proteins, and a specific deactivator of potentially toxic electrophilic agents. A number of compounds have been developed to alter the metabolism and intracellular concentration of GSH. The intracellular GSH levels in different cell systems can be decreased after treatment with diethylmaleate (DEM),phorone (diisopropylidene acetone), and D,Lbuthionine-S-R-sulfoximine(BSO) (Meister, '85). Teratogenicity of the antitumor agent 5fluorouracil (5-FU) has been characterized by inducing limb malformations in mice (Dagg, '60; Dagg et al., '66; Katagiri, '83). There is a considerable advantage in using limb malformations to evaluate the protective and/or augmentative effect of chemicals on teratogenicity, because severity of limb malformations can be easily defined by the number of missing digits. We have previ-
0 1990 WILEY-LISS, INC.
ously shown that GSH depletion with DEM increases the incidence of oligodactyly; on the contrary, pretreatment with GSH decrease the incidence of oligodactyly induced with 5-FU in mice (Naya et al., '87). DEM, however, inhibited microsomal p-450-dependent enzymes, as well as depleting GSH, thus interfering with the bioactivation of halogenated hydrocarbons (Anders, '78; Suarez et al., '81).Therefore, DEM may deplete endogenous GSH nonspecifically. On the other hand, phorone depletes GSH but does not interfere with the microsomal mixed-function oxidase system (Siegers et al., '85). Recently, BSO, the specific inhibitor of y-glutamyl cysteine synthetase, has been used extensively to deplete cellular GSH in studies designed to enhance the toxic effects of various antitumor agents (Arrick and Nathan, '84; Russo et al., '86). In the present study, we used phorone and BSO in order to investigate the relationship of GSH levels to 5-FU teratogenicity in mice. Received June 12, 1989; accepted September 18, 1989
276
M. NAYA ET AL 2o
r
T
L
Phorone
-
C P h o r o n e t BSO
** I
OL I
0
I 2
I 4
I 6
!c-------I 24
Hours a f t e r t r e a t m e n t Fig. 1. Effects of phorone and/or buthionine sulfoximine (BSO) on glutathione (GSH) levels in maternal serum of ICR mice. Phorone 250 mg/kg i.p., BSO 200 mgikg i.p., or phorone 125 mgikg plus BSO 100 mgikg i.p. was administered on day 11 of gestation. Pregnant mice were killed a t 0, 2, 4,6, or 24 hours after administration of phorone and/or BSO. Levels of GSH were determined by high performance liquid chromatography. Each value represents the mean i S.D. of four animals. *P
