Cong. Anom., 28: 15 7 16 7, 1988 Original Effects of Antimutagens on the Teratogenicity of N-Methyl­ N'-Nitro-N-Nitrosoguanidine in Mice Yasunobu MORITA and Masahiro MIZUTANI Hatano Research Institute, Food and Drug Safety C'rntcr, Ochiai, Hadano, Kana­ gawa 257, Japan ABSTRACT We examined whether vanillin (VA) and CoC'l 2 -6H 2 0(CoC1 2 ), anti­ mutagens, which have mutation suppressing effect, i.e., promotion of cellular repair function in vitro, can modify the teratogenicity in mice caused by N-methyl­ N'-nitro-N-nitrosoguanidine (MNNG), a direct-acting monofunctional alkylating agent. ICR mice were treated with MNNG alone (single IP dose of 40 or 60 mg/kg) or in combination with the antimutagen on day 11 of gestation. Embryotoxicity and teratogenicity were examined at term. The incidence of MNNG-induced syndactyly in the fore- and hindlimbs was significantly decreased by VA (50 mg/kg, IP) or CoC1 2 (IO mg/kg, IV) and a tendency to decrease in the incidence of oligodactyly was noted as well. On the other hand, the incidence of MNNG-induced brachy­ dactyly was increased by VA or CoC1 2 • Though the mechanism of the modifying effects of both VA and CoC'l 2 on MNNG-induced malformations could not be delineated in the present study, the results indicate that the antimutagens which stimulate DNA recombination repair in vitro modify the manifestation of malformations caused by teratogens that attack the fetal DNA in the initial teratogenic mechanism. Key words: N-methyl-N'-nitro-N-nitrosoguanidine, antirnutagens, vanillin, CoCl 2 , fetal development, malformations, modification, mice N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) is a direct-acting alkylating agent that possesses mutagenic activity (Mandell and Greenberg, 1960; Hsu et al., 1977) and carcinogenic activity (Sugi­ rnura et al., 1966 ). Further study by Inouye and Murakami (I 978) has demonstrated that this compound is also teratogenic in mice. Maternal intraperitoneal administration of 60 mg/kg of MNNG on day 11 of gestation in mice induced brain, facial, vertebral, rib and limb malformations in the offspring. Digital defects were induced also at a higher incidence (Manson and Marian, 1983). At this dose (60 mg/kg), MNNG induced less lethal damage to the fetus compared to fetal mortality caused by other alkylating agents Received February 3, 1988 i"iilll*'f,t, Jkti-iE"(L (11-t Ht,\/,'l,\/,'ii:·'t'-t:;,, ?-40HJf'}):riJr, 9'257 4-f'l'frfi{f;{:-n\) :i 158 Y. Morita and M. Mizutani such as MMS (t11ethyl methanesulfonate). Recently. antimutagens capable of suppressing cellular mutagenesis in the bacterial system have hcen reported ( Kada and Kanematsu, 1978 ). Among those antimutagens cobaltous chloride (CoCl 2 • <,11 2 0) ( Kiuchi ct al.. I 984) and vanillin (Ohta ct al., 1986) are categorized as bio-antimutagens that suppress t11utagcncsis by promoting cellular repair function after inducing primary DNA damage (Kada ct al.. 1986). MNNG treatment (0.25 µg/ml) of cultured FM3A cells induced mutation result­ ing in 6-thioguaninc resistant clones. When CoCl 2 -6H 2 0 was present (at a concentration of 1.0 µg/ml) in the culture medium of FM3A cells during the whole mutation expression time, after treat­ t11cnt with MNNG for 2 hours. the frequency of 6-thioguanine resistant clones was slightly reduced ( Unpublished data by authors). More recently. it has been reported that post-treatment of vanillin reduced the formation of hrcakagc-type chromosome aherrations of mitomycin C (MMC)-trcated Chinese hamster ovary cells. despite the increase in frequency of MMC-induced SC Es (Sasaki et al., 1987). It is generally accepted that mutagenicity and carcinogenicity arc highly correlated with each other in terms of short-term assay for carcinogenicity (Kawachi et al., 1980). However there is no evident correlation between either of these two forms of toxicity and teratogcnicity due to much more complex factors involved in the initial process of abnormal development. Nevertheless, alkylat­ ing agents found to be both mutagcnic and carcinogenic have also been proven to be teratogenic when appropriately tested (Wilson, 1972). As for the teratogenic mechanism. especially of alkylating agents, it has been proposed that genetically transmitted mutational mechanisms not involving cell death can profoundly influence the course of morphogenesis. although cell death plays an important role in the occurrence of birth defects (Manson, 1981; Neubert, 1980; Platzck et al., 1982). In this study, we examined whether substances which have been shown to be antimutagenic in vitro (e.g., CoCJ 2 -6H 2 O and vanillin) can modify the tcratogenicity caused by MNNG in mice. MATERIALS AND METHODS The animals used in this study were Sic: ICR mice purchased from Shizuoka Agricultural Co­ operative Association for Laboratory Animals. The mice were housed in plastic cages with wood chip bedding and kept in a temperature (24±1 °C) - and humidity (55±5 %) - controlled room with a fixed 12-hour lighting schedule throughout the year. Food (laboratory chow CA-I, CLEA Japan, Inc.) and water were allowed ad libitum. Estrous females (9-13 weeks old) were housed overnight with males in pairs. The next morning, female mice with copulation plugs were designated to be on day O of gestation. Treatment: To determine the effect of antimutagens on fetal development, mice on day 11 of ges­ tation at noon were separately given a single intraperitoneal dose of 50 mg/kg of vanillin (VA, Tokyo Kasei Industries. Inc.) or single intravenous dose of 10 mg/kg of CoCl 2 -6H2 O(CoC1 2 , Wako Pure Chemical Industries. Inc.). In another experiment, 2 groups of mice on day 11 of gestation (at noon) were given intraperi­ toneal dose of 40 mg/kg and 60 mg/kg of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, Aldrich Chemical Company. Inc.). respectively. Each group of MNNG-treated mice was divided into 2 sub- Effects of antimutagcns on the teratogenicity of MNNG 159 groups. A single dose (50 mg/kg, IP) of VA was administered to one sub-group (MNNG+V A group) and CoC1 2 (IO mg/kg, IV) to the other sub-group (MNNG+CoC1 2 group) one hour after MNNG treatment. Saline was used as diluent for MNNG, VA and CoC1 2 • Control groups for this study were set up as follows: one control group was administered saline only (NSS, 10 ml/kg, IP) and the other control group was given saline (same dose, IP) one hour after MNNG treatment (MNNG+saline group). Observations: On day 18 of gestation, the dams were humanely killed and the number of im­ plants, resorptions, dead fetuses and live fetuses were counted. Live fetuses were weighed and ex­ amined for external malformations under a dissecting microscope, and then cleared and stained by means of Dawson's technique (Dawson, 1926) for skeletal examination. Furthermore, for examina­ tion of the phalanges stained cartilagious and ossified components in the fore- and hindlimbs, some of the specimens stained with alizarin red S were subsequently stained with toluidine blue according to the modified method of Burdi (Burdi, 1965). Statistics: Data obtained herein were calculated on the basis of the litters and analyzed by means of the following statistical methods. Fetal body weight and number of live fetuses were analyzed using student's t-test. Fetal mortality and incidence of fetal malformations and variations were analyzed using Wilcoxon's rank sum test. RESULTS The effects of the· antimutagens, VA and CoC1 2 , on fetal development in mice are shown in Table I. Effects of VA and CoCl2 were comparable to that of the saline control with respect to the number of live fetuses, fetal body weight, fetal mortality and incidence of external and skeletal abnormalities. The influence of these antimutagens on MNNG-induced fetal defects is shown in Table 2. Body weight of fetuses from dams to which doses of 60 mg/kg of MNNG had been administered showed very considerable reduction with or without the antimutagens. Cleft palate and digital malformations of the fore- and hindlimbs were observed with a correlation to dose rate of MNNG. At a dose of 60 mg/kg of MNNG, these malformations appeared in a high frequency. As a brain malformation, flatness at the top of the head was noted in fetuses of all treated groups following the administration of 60 mg/kg of MNNG. This malformation appeared to correspond to microcephaly. The incidence of external malformations in MNNG+VA group was comparable to that in MNNG+saline group. The incidence of microcephaly and facial defects (cleft palate, microtia etc.), however, showed a tendency to decrease compared to MNNG (60 mg/kg)+saline group. In MNNG (60 mg/kg)+CoCl2 group, fetuses with external malformations were less than those in MNNG (60 mg/kg)+saline group. As for individual malformations, the incidence of microcephaly and facial defects (cleft palate, microtia etc.) was significantly decreased and that of digital malformations in the fore- and hindlimbs showed a tendency to decrease compared to MNNG (60 mg/kg)+saline group. Fig. 1 shows the influence of VA and CoCl2 on the incidence of MNNG-induced cleft palate and digital malformations (oligodactyly, brachydactyly and syndactyly) in the fore- and hindlimbs. The incidence of syndactyly in the fore- and hindlimbs was significantly decreased both in MNNG (60 mg/kg)+ VA or MNNG (60 mg/kg)+ CoCl2 groups, with a decrease in oligodactyly also being 160 Y. Morita and M. Mizutani Table l Influence of vanillin and CoC1 1 on fetal development in mice External abnormalities (% )a Skeletal abnormalities (%)a No. Mean no. Fetal Fetal Ex- Treat- of of live body mortal- ternal a ment litters fetuses weighta ity abn. Brain Face Others Malfor- Variationse (g) (%)a (%)a mations Saline 8 13.6 1.34 5.1 0 0 0 0 l.Oc 34.4 ± 0.6 ±0.06 ±1.8 ±l.0 ± 6.0 VA 3 13.7 1.42 9.6 0 0 0 0 0 25.7 so ± 1.8 ±0.06 ±4.8 ± 17.4 mg/kg 1.6b CoCl 1 4 12.8 1.39 4.6 1.6 0 0 4_9d 51.4 10 ± 2.7 ±0.05 ± 1.6 ±1.6 ± 1.6 0 ±l.1 ± I 2.5 mg/kg Vanillin (VA) or CoC11 was administered on day 11 of gestation.