Hideo Tezuka

Suppression of Cell Proliferation by Interferon-Alpha through Interleukin-1 Production in Adult Rat Dentate Gyrus

The therapeutic use of interferon-alpha (IFN-α), a proinflammatory cytokine, is known to cause various neuropsychiatric adverse effects. In particular, depression occurs in 30–45% of patients, frequently interrupting treatment. IFN-α-treated animals also show depression-like behaviors. However, mechanisms underlying the depression caused by IFN-α remain to be defined. Recently, a decrease in adult hippocampal neurogenesis was revealed as a possible neuropathological mechanism of depression. Therefore, we investigated the effect of subchronic IFN-α treatment on neurogenesis in the adult rat dentate gyrus (DG). Immediately after the administration of IFN-α for 1 week, a decrease in the number of 5-bromo-deoxyuridine-labeled proliferating cells was observed in the DG; however, no effect was detected on the expression of mature neuronal phenotype in the newly formed cells 3 weeks later. Also, an increase in the level of interleukin-1beta (IL-1β), a major proinflammatory cytokine, was observed in the hippocampus following the administration of IFN-α. Furthermore, coadministration of an IL-1 receptor antagonist completely blocked the IFN-α-induced suppression of the cell-proliferative activity in the DG. Our results indicate that IFN-α suppresses neurogenesis in the DG, and that IL-1β plays an essential role in the suppression. The decreased cell proliferation caused by IFN-α-induced IL-1β may be responsible, at least in part, for IFN-α-induced depression.

Mutagenicity of Pesticides

Microbial mutagenicity screening studies employing Ames tests were done on 228 pesticides. As a result, 50 compounds showed mutagenicity and five of them required metabolic activation for their activities. Among the various compound groups, organic phosphates, halogenated alkanes and dithiocarbamates contained mutagens at a higher ratio. Mutagenicity of these pesticides was less potent than that of other mutagens. Cytogenetic studies on the pesticides which were positive in microbial assays revealed a good correlation between the ability to induce sister chromatid exchanges or chromosomal aberrations and the mutagenic potency in bacteria. Dominant lethal studies on 1, 2-dibromo-3-chloropropane (DBCP) and ethylene dibromide (EDB) disclosed that DBCP gave positive results in rats but not in mice, although EDB was negative in both species. DBCP was also positive in a sex-linked recessive lethal test using Drosophila melanogaster. Occupational exposure to pesticides, especially gaseous ones like DBCP, in industry or in agriculture seems hazardous from the genetic toxicological point of view.

Avoiding the effect of linked genes is crucial to elucidate the role of Apcs in autoimmunity.

To the editor: It was reported that mice with targeted disruption of the gene that encodes serum amyloid P-component (Sap, encoded by Apcs) with a mixed genetic background (129/Sv × C57BL/6; F2) developed high titers of antibody to nuclear antigens (ANA) and severe glomerulonephritis, a phenotype resembling systemic lupus erythematosus (SLE)1. Thus, it was speculated that Sap contributes to the pathogenesis of SLE. But the Apcs gene on mouse chromosome 1 is located in a region harboring several genes reported to be associated with SLE susceptibility in multiple strain combinations2. This suggests that ignoring the confounding effect of genes around the disrupted Apcs gene in the knockout mice might lead to misinterpretation of the role of Sap in autoimmunity. To assess the effects of genetic background and genes linked to the disrupted Apcs gene, we first transferred the disrupted Apcs gene from Apcs–/– mice generated from 129/Sv//Evderived embryonic stem cells and backcrossed into C57BL/6 (129/Sv//Ev × C57BL/6; F4)3,4 to C3H/He mice as follows: a male Apcs–/– 129/ Sv//Ev × C57BL/6 mouse was mated with C3H/He female mice. Homozygous mutant (Apcs–/–) and wild-type (Apcs+/+) F2 mice from the heterozygote (Apcs+/–; F1) matings were analyzed. There was no significant difference in the titers of ANA between 12–13month-old Apcs–/– (n = 50; 27 females and 23 males) and Apcs+/+ (n = 41; 18 females and 23 males) 129/Sv//Ev × C57BL/6 × C3H/He littermates (Fig. 1a). The result indicates that Sap deficiency in itself does not promote autoimmunity. We then crossed the Apcs–/– 129/ Sv//Ev × C57BL/6 mice back into C57BL/6 for 10 generations to attain genetic homogeneity. The backcrossed 11–14-month-old (n = 37; 21 females and 16 males) Apcs–/– mice produced significantly higher titers of ANA than did Apcs+/+ (n = 29; 19 females, P = 0.00001, and 10 males, P = 0.0008) littermates (Fig. 1b). A linkage study indicated that the backcrossed Apcs–/–mice still carried at least a 6.2-cM 129/ Sv//Ev-type chromosome segment marked by D1Mit149, D1Mit206, D1Mit456 and Apcs, between D1Mit36 and D1Mit115. The segment overlaps the mouse SLE susceptibility loci Sle1 and Nba2 and harbors genes implicated in SLE susceptibility, such as Fcgr2b and interferonactivated gene 202 (Ifi202)2,5. Mice congenic for the Nba2 locus with a C57BL/6 genetic background (C57BL/6. Nba2) had increased levels of Ifi202 mRNA and protein in their spleen cells, which correlated with lupus susceptibility5. Similar to the Apcs–/– 129/Sv//Ev × C57BL/6 mice, the C57BL/6.Nba2 mice develop substantially higher titers of ANA relative to C57BL/6 mice but they never develop severe glomerulonephritis4,5. Thus, we compared the level of Ifi202 mRNA in the spleen of Apcs–/– and Apcs+/+ 129/Sv//Ev × C57BL/6 littermates by northern blot hybridization. A 1.8-kilobase Ifi202 mRNA was detected in the Apcs–/– but not in the Apcs+/+ mice (Fig. 2a). Because the expression of Ifi202 is inducible by lipopolysaccharide (LPS), we compared the levels of Ifi202 mRNA in the spleen of Apcs–/–, Apcs+/– and Apcs+/+ mice at 4 h after intraperitoneal injection of Salmonella typhimirium LPS (15 mg/kg body weight). The levels of splenic Ifi202 mRNA of LPS-injected Apcs–/– and Apcs+/– mice were substantially higher than those of Apcs+/+ littermates and the Ifi202 mRNA level of Apcs–/– mice was approximately twice as high as that of Apcs+/– mice (Fig. 2a). Because transcription of Ifi202 is reportedly impaired in the C57BL/6 mouse5, the data suggest that the expression of the Ifi202 gene linked to the C57BL/6-derived wild-type Apcs gene is impaired but that linked to the 129/Sv//Ev-derived disrupted Apcs gene is not. The levels of splenic Ifi202 mRNA of wild-type 129/Sv//Ev and C3H/He mice are much the same and are substantially higher than those of wild-type C57BL/6 mice. Because the disrupted and wild-type Apcs alleles in the 129/Sv//Ev × C57BL/6 × C3H/He mice are derived from 129/Sv//Ev and C3H/He mice, respectively, the level of expression of the Apcs-linked Ifi202 gene between Apcs–/– and Apcs+/+ 129/Sv//Ev × C57BL/6 × C3H/He mice is expected to be much the same. Indeed, no differences were detected in the Ifi202 mRNA levels between the Apcs–/– and Apcs+/+ mice as shown in Fig. 2b. Thus, differences in mean levels of ANA between the Apcs–/– and Apcs+/+ mice correlate with differences in the levels of splenic Ifi202 mRNA independent of Sap: no difference in the titers of ANA between the Apcs–/– and Apcs+/+ 129/Sv//Ev × C57BL/6 × C3H/He mice was detected, whereas the Apcs–/– 129/Sv//Ev × C57BL/6 mice produced significantly higher titers of ANA than control Apcs+/+ littermates (Fig. 1a,b). A N A ti te r 2,560 2,560

Milk Inhibits the Regression of 7,12-Dimethylbenz(a)anthracene-Induced Mammary Tumors in Ovariectomized Rats

Epidemiological studies have yielded inconsistent results regarding the relation between the milk consumption and breast cancer risk. In this study, rats were induced mammary tumors by 7,12-dimethylbenz(a)anthracene. When tumors developed to acceptable levels, rats were placed into 1 of 3 treatment groups. Those in the negative control group and the milk group were ovariectomized, whereas those in the positive control group were sham operated. After grouping, tumor incidence remained 100%, and tumor number and volume increased in the positive control group. However, tumors in the 2 ovariectomized groups regressed. Compared with the negative control group, tumor incidence and tumor number and volume per rat in the milk group became significantly higher from Week 6 and Week 4, respectively. Insulin-like growth factor-I levels were borderline significantly higher in the milk group than in the negative control group at autopsy. Although plasma 17β-estradiol levels did not differ significantly, estrogenicity was found in the milk group because uterine weight was significantly heavier in the milk group than in the negative control group. In conclusion, commercial milk inhibited the regression of carcinogen-induced mammary tumors in ovariectomized rats.

Heritable translocation study in male mice with trimethyl phosphate

Dominant lethal and heritable translocation studies were performed in male mice receiving a single intraperitoneal injection of trimethyl phosphate (TMP). The germ cell stage investigated was the spermatid. Methyl methanesulfonate (MMS) was used as a positive control in the latter study. A dominant lethal assay gave marked dose-dependent increases in early fetal deaths. Heritable translocations were detected at 1000 or 1500 mg of TMP/kg in F1 male progeny when screening for semi-sterility and cytogenetically analyzing the meiotic or mitotic chromosomes. Translocation induction was higher at the higher TMP dose (14.3%) than at the lower dose (5.3%) and the yield from the higher dose was similar to that induced by 50 mg of MMS/kg (11.0%). Most of the translocation carriers were semi-sterile or sterile. The data confirm conclusions from other dominant lethal studies showing TMP to be capable of causing chromosomal damage in mouse spermatids and show that certain types of damage result in heritable translocations.