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A newly discovered metabotropic glutamate receptor (mGluR) 1 allosteric antagonist, 2-cyclopropyl-5-[1-(2-fluoro-3-pyridinyl)-5-methyl-1H-1,2,3-triazol-4-yl]-2,3-dihydro-1H-isoindol-1-one (CFMTI), was tested both in vitro and in vivo for its pharmacological effects. CFMTI demonstrated potent and selective antagonistic activity on mGluR1 in vitro and in vivo after oral administration. CFMTI inhibited l-glutamate-induced intracellular Ca2+mobilization in Chinese hamster ovary cells expressing human and rat mGluR1a, with IC50values of 2.6 and 2.3 nM, respectively. The selectivity of CFMTI to mGluR1 over mGluR5 was >2000-fold, and CFMTI at 10 μM showed no agonistic or antagonistic activities toward other mGluR subtypes and other receptors. It antagonized face-washing behavior in mice induced by (S)-3,5-dihidroxyphenylglycine at a dose range of 3 to 30 mg/kg, for which receptor occupancy was 73 to 94%. As with the classical neuroleptic haloperidol and an atypical antipsychotic, clozapine, orally administered CFMTI reduced methamphetamine-induced hyperlocomotion and disruption of prepulse inhibition (PPI) at the same dose range as required to antagonize the face-washing behavior. CFMTI and clozapine improved ketamine-induced hyperlocomotion, PPI disruption and (5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801)-induced social withdrawal without any cataleptogenic activities, whereas haloperidol only improved ketamine-induced hyperlocomotion. CFMTI, unlike clozapine, caused neither hypolocomotion nor motor incoordination at therapeutic doses. In c-fos expression studies, CFMTI and clozapine increased the number of fos-positive neurons in the nucleus accumbens and medial prefrontal cortex but not in the dorsolateral striatum. These results suggest that the antipsychotic activities of mGluR1 antagonists are more similar to those of atypical antipsychotics than those of typical antipsychotics.

A newly discovered metabotropic glutamate receptor (mGluR) 1 allosteric antagonist, 2-cyclopropyl-5-[1-(2-fluoro-3-pyridinyl)-5-methyl-1H-1,2,3-triazol-4-yl]-2,3-dihydro-1H-isoindol-1-one (CFMTI), was tested both in vitro and in vivo for its pharmacological effects. CFMTI demonstrated potent and selective antagonistic activity on mGluR1 in vitro and in vivo after oral administration. CFMTI inhibited L-glutamate-induced intracellular Ca(2+) mobilization in Chinese hamster ovary cells expressing human and rat mGluR1a, with IC(50) values of 2.6 and 2.3 nM, respectively. The selectivity of CFMTI to mGluR1 over mGluR5 was >2000-fold, and CFMTI at 10 microM showed no agonistic or antagonistic activities toward other mGluR subtypes and other receptors. It antagonized face-washing behavior in mice induced by (S)-3,5-dihidroxyphenylglycine at a dose range of 3 to 30 mg/kg, for which receptor occupancy was 73 to 94%. As with the classical neuroleptic haloperidol and an atypical antipsychotic, clozapine, orally administered CFMTI reduced methamphetamine-induced hyperlocomotion and disruption of prepulse inhibition (PPI) at the same dose range as required to antagonize the face-washing behavior. CFMTI and clozapine improved ketamine-induced hyperlocomotion, PPI disruption and (5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801)-induced social withdrawal without any cataleptogenic activities, whereas haloperidol only improved ketamine-induced hyperlocomotion. CFMTI, unlike clozapine, caused neither hypolocomotion nor motor incoordination at therapeutic doses. In c-fos expression studies, CFMTI and clozapine increased the number of fos-positive neurons in the nucleus accumbens and medial prefrontal cortex but not in the dorsolateral striatum. These results suggest that the antipsychotic activities of mGluR1 antagonists are more similar to those of atypical antipsychotics than those of typical antipsychotics.

The aim of this study was to clarify the relationship between receptor occupancy and In vivo pharmacological activity of mGluR1 antagonists. The tritiated mGluR1-selective allosteric antagonist [3H]FTIDC (4-[1-(2-fluoropyridin-3-yl)-5-methyl-1H-1,2,3-triazol-4-yl]-N-isopropyl-N-methyl-3,6-dihydropyridine-1(2H)-carboxamide) was identified as a radioligand having high affinity for mGluR1-expressing CHO cells (KD= 2.1 nM) and mouse cerebellum (KD= 3.7 nM). [3H]FTIDC bound to mGluR1 was displaced by structurally unrelated allosteric antagonists, suggesting there is a mutual binding pocket shared with different allosteric antagonists. The binding specificity of [3H]FTIDC for mGluR1 in brain sections was demonstrated by the lack of significant binding to brain sections prepared from mGluR1-knockout mice. Ex vivo receptor occupancy with [3H]FTIDC revealed that the receptor occupancy level by FTIDC correlated well with FTIDC dosage and plasma concentration. Intracerebroventricular administration of (S)-3,5-dihydroxyphenylglycine is known to elicit face washing behavior that is mainly mediated by mGluR1. Inhibition of this behavioral change by FTIDC correlated with the receptor occupancy level of mGluR1 in the brain. A linear relationship between the receptor occupancy and In vivo activity was also demonstrated using structurally diverse mGluR1 antagonists. The receptor occupancy assays could help provide guidelines for selecting appropriate doses of allosteric mGluR1 antagonist for examining the function of mGluR1 In vivo.

We have recently shown that immunodeficient (SCID) mice, which lack functional T and B cells, are highly susceptible to low dose site specific induction of colon aberrant crypt foci (ACF), surrogates for colon tumors, by 2-amino-3-methylimidazo[4,5-f ]quinoline (IQ). To test whether long-term exposure to a high dose in the diet might prove carcinogenic to the SCID mouse colon, in contrast to other mice strains tested to date, the compound was administered at 300 p.p.m. in the diet to female 6–7-week-old SCID mice for 32 weeks. IQ induced high numbers of ACF, hyperplastic polyps, dysplasia, and colon adenomas, as well as hepatocellular altered foci and liver adenomas. Induction of colon tumors did not correlate with the main sites where ACF developed, the proximal colon, however, being seen mainly in the mid and distal colon. Induction of colon tumors correlated significantly with the incidence of dysplasia, crypt height, the mitotic index, cell proliferation and numbers of 8-hydroxydeoxyguanosine (8-OHdG)-positive cells in the colon crypt, particularly in mid and distal colon. Administration of 20% ωω-6 polyunsaturated fatty acids (corn oil), ωω-3 polyunsaturated fatty acids (perilla oil), or monounsaturated fatty acids (olive oil) simultaneously with IQ in the diet resulted in: (i) inhibition of colon and liver tumor induction by corn and perilla oil, whereas olive oil showed no effects; (ii) no reduction in total numbers of ACF by corn oil or perilla oil but significant suppression in the olive oil treated group; (iii) inhibition of tumor development particularly by ωω-3 polyunsaturated fatty acids in perilla oil, correlating significantly with decreased cell proliferation in both colon and liver and a marked decrease in crypt heights and mitotic indices. Selective reduction in the numbers of 8-OHdG-positive nuclei, mainly in the middle and distal colon crypts, was also found to correlate with tumor inhibition. Thus, the results indicate carcinogenicity of IQ in the colon of the SCID mouse and preventive effects of polyunsaturated fatty acids.

We have recently shown that immunodeficient (SCID) mice, which lack functional T and B cells, are highly susceptible to low dose site specific induction of colon aberrant crypt foci (ACF), surrogates for colon tumors, by 2-amino-3-methylimidazo[4,5-f ]quinoline (IQ). To test whether long-term exposure to a high dose in the diet might prove carcinogenic to the SCID mouse colon, in contrast to other mice strains tested to date, the compound was administered at 300 p.p.m. in the diet to female 6-7-week-old SCID mice for 32 weeks. IQ induced high numbers of ACF, hyperplastic polyps, dysplasia, and colon adenomas, as well as hepatocellular altered foci and liver adenomas. Induction of colon tumors did not correlate with the main sites where ACF developed, the proximal colon, however, being seen mainly in the mid and distal colon. Induction of colon tumors correlated significantly with the incidence of dysplasia, crypt height, the mitotic index, cell proliferation and numbers of 8-hydroxydeoxyguanosine (8-OHdG)-positive cells in the colon crypt, particularly in mid and distal colon. Administration of 20% omega-6 polyunsaturated fatty acids (corn oil), omega-3 polyunsaturated fatty acids (perilla oil), or monounsaturated fatty acids (olive oil) simultaneously with IQ in the diet resulted in: (i) inhibition of colon and liver tumor induction by corn and perilla oil, whereas olive oil showed no effects; (ii) no reduction in total numbers of ACF by corn oil or perilla oil but significant suppression in the olive oil treated group; (iii) inhibition of tumor development particularly by omega-3 polyunsaturated fatty acids in perilla oil, correlating significantly with decreased cell proliferation in both colon and liver and a marked decrease in crypt heights and mitotic indices. Selective reduction in the numbers of 8-OHdG-positive nuclei, mainly in the middle and distal colon crypts, was also found to correlate with tumor inhibition. Thus, the results indicate carcinogenicity of IQ in the colon of the SCID mouse and preventive effects of polyunsaturated fatty acids.

We previously reported p53mutations to be frequent (greater than 70%), whereas both H‐rasmutations and microsatellite instability (MSI) were infrequent (about 10%), in urinary bladder carcinomas (UBCs) and their metastatic foci in the N‐butyl‐N‐(4‐hydroxybutyl)nitrosamine (BBN)‐induced mouse urothelial carcinogenesis model. In the present study, an analysis of p53and H‐rasmutations as well as MSI was performed on 12 renal pelvic carcinomas (RPCs) and 8 metastatic or invading foci produced by the same experimental procedure. Histologically, 10 of the RPCs were transitional cell carcinomas and the remaining 2 were squamous cell carcinomas. p53mutations were infrequent and only found in one primary RFC (8%), its metastatic foci and an invading lesion in another animal (in a total 2 of 12; 17%). H‐rasmutations were slightly more frequent (found in 3 of 12 animals; 25%), 4 of 5 involving codon 44, GTG to GCG, not a hot‐spot reported for human cancers. In two cases, H‐rasmutations were confined to lung metastasis and not detectable in their primary RPCs. MSI analysis was available for 6 pairs of primary RPCs and their metastatic foci, and 4 animals (67%) had MSI at one or more microsatellite loci. Overall, the distribution of genetic alterations differed from that in UBCs produced by the same experimental protocol. The results thus suggest that different genetic pathways may participate in carcinogenesis of the upper and lower urinary tract due to BBN.

Abstract: Treatment of C57BL/6 J (B6) and NON male mice with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) resulted in a high incidence of bladder cancer. The mean survival period, however, differed significantly by strain: 481  219 days in B6 (n = 31) and 203  119 days in NON (n = 30) (P < 0.0001). Major causes of death were renal failure due to obstruction of the urinary tract, or local invasion of tumors. The fact that the BBN-treated NON  B6 reciprocal F1 mice had survival periods as short as those of the parental NON mice suggests a genetically dominant susceptibility in NON or recessive resistance in B6. A linkage analysis of 248 back-cross mice to B6 suggested at least two quantitative trait loci determining the length of the survival period: one was mapped close to D2Mit260 (logarithm of odds, LOD, score 2.21), a microsatellite marker locus 83 cM from the centromere on chromosome 2, and another was close to D6Mit159, 7 cM from the centromere on chromosome 6 (LOD score 2.51).

In our two‐stage model of rat urinary bladder carcinogenesis employing N‐butyl‐N‐(4‐hydroxybutyl)nitrosamine (BBN) as the initiator, sodium L‐ascorbate (Na‐AsA) exhibits dose‐dependent promotion. In the present study, in order to assess the possible reversibility of the promoting effects, we investigated how different administration periods of Na‐AsA influence its promoting activity. In experiment 1, rats were treated with 5% Na‐AsA for different administration periods with or without withdrawal and injected with 5‐bromo‐2′‐deoxyuridine (BrdU) to allow determination of the cell proliferation status. Replicative DNA synthesis in the urinary bladder epithelium was shown to return to normal after removal of the promoting stimulus. In experiment 2, rats were initially given BBN for 4 weeks and subsequently received 16 weeks of Na‐AsA, alternating with basal diet, at intervals of 4, 8 or 16 weeks, within total 32‐week period. The longer the continuous exposure to Na‐AsA, the greater the yield of papillomas and carcinomas in the urinary bladder. In experiment 3, Na‐AsA was given for 4 or 8 weeks after BBN initiation and the animals were killed at weeks 8 and 12. Both promotion of lesion development and increase of DNA synthesis in the urinary bladder epithelium were dependent on the length of exposure to Na‐AsA and the total period of exposure. The results indicate that the promoting effects of Na‐AsA in urinary bladder carcinogenesis are reversible to certain extent after its withdrawal, and the existence of cumulative exposure time threshold seems likely.

We attempted to establish an animal model for renal pelvic and ureteral carcinomas. In Experiment 1, Sprague-Dawley (SD)/cShi and SD/gShi strains of male and female rats and Fischer-344 (F-344) DuCrj and LEW/Crj strains of male rats, 6 wk old, were given 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) for 12 wk and then treated without chemical for 10 wk. Only the SD/cShi strain. which has spontaneous hydronephrosis and hydroureter, showed high incidences of carcinomas in the renal pelvises and ureters, whereas the other strains did not. In Experiment 2, SD/cShi and Crj:CD strains of male and female rats. 6 wk old, were exposed to 0.05% BBN for 20 wk. Both sexes of SD/cShi rats had high incidences of bilateral carcinomas in renal pelvises and/or ureters associated with urinary bladder carcinomas, whereas both sexes of Crj:CD rats had urinary bladder tumors only. These results indicate that the SD/cShi strain may be a suitable animal for the induction of renal pelvic and ureteral carcinomas that appear to depend on urine stagnation secondary to hydronephrosis and hydroureter.

O⁶-methylguanine is known as one of the major premutagenic lesions in the human and rodent carcinogenesis process. O⁶-methylguanine-DNA methyltransferase (MGMT), which repairs methylated guanine bases, might prevent the G:C to A:T transition, and transgenic mice carrying this MGMT gene have been reported to be less sensitive to the carcinogenicity of certain alkylating agents. Here we utilized MGMT transgenic mice to assess the significance of O⁶-methylguanine formation during urinary bladder carcinogenesis. In experiment 1, 100 and 60 ppm N-butyl-N-(4-hydroxybutyl)nitrosamine was given for 20 weeks to transgenic and non-transgenic mice in their drinking water. The incidences of urinary bladder carcinomas were not different between transgenic mice and non-transgenic mice. The mutational spectrum of the p53 gene was evaluated by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis and direct sequencing. The pattern of p53 mutations of transgenic and non-transgenic mice did not differ, and the frequencies of mutations were 40% and 42%, respectively. G:C to A:T transition mutations were particularly infrequent (1 of 14 mutations, 7%). In experiment 2, N-methyl-N-nitrosourea, which might induce O⁶-methylguanine in affected alleles, was given once a week, 3 times (total 5 mg) by direct instillation into the urinary bladder through an abdominal incision. No significant neoplastic lesions were detected, although the experiment was limited by severe toxicity of the treatment. p53 immunostaining was done and there was no difference in transgenic and non-transgenic mice. These results suggest that O⁶-methylguanine formation might not be a significant mutational factor in these mouse urinary bladder carcinogenesis models. Teratogenesis Carcinog. Mutagen. 18:101–110, 1998. © 1998 Wiley-Liss, Inc.

Our previous data showed that F344/DuCrj and LEW/Crj rat strains bearing the type a catalase-1 locus (CS1^a) are sensitive to the promoting activity of sodium L-ascorbate (Na-AsA) in 2-stage urinary bladder carcinogenesis, whereas ODS/Shi and WS/Shi rat strains bearing the type b catalase-1 locus (CS1^b) are resistant. In present study, we investigated the susceptibility of F344/Shi rats also bearing the CS1 to the Na-AsA-promoting effects on bladder tumor development. Male rats, 6 weeks old, were given 0.05% N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) in their drinking water for 4 weeks, then fed either basal diet supplemented with 5% Na-AsA or no chemicals for 32 weeks. The rats given BBN alone had a few small carcinomas in the urinary bladder. In contrast, animals administered BBN-Na-AsA had many large carcinomas. Administration of Na-AsA was associated with significant elevation of urinary pH and L-ascorbic acid. The results indicate that F344/Shi rats are sensitive to the promoting effects of Na-AsA on 2-stage urinary bladder carcinogenesis, and thus that the CS1 locus may not influence susceptibility to promotion. Teratogenesis Carcinog. Mutagen. 18:27–33, 1998. © 1998 Wiley-Liss, Inc.

Rat strain differences in sensitivity to the promoting effect of sodium L‐ascorbate (SA) on the development of urinary bladder tumors were investigated. In experiment 1, WS/Shi (WS), ODS/Shi‐od/od (ODS), and LEW/Crj (LEW) rats were initiated with 0.05%N‐butyl‐N‐(4‐hydroxybutyl)‐nitrosamine (BBN) in their drinking water and subsequently given basal Oriental MF diet (M) with or without a 5% SA supplement. In LEW rats the SA treatment increased the induction of neoplastic lesions in the urinary bladder, whereas WS and ODS animals proved unresponsive to its promoting effects. In experiment 2, WS and F344 rats were maintained on two kinds of commercial basal diets, M and CLEA CA‐1 (C), during administration of SA, since dietary factors can influence promoting effects. Feeding M during the promotion period in F344 rats yielded significantly more neoplastic lesions than feeding C, but in WS rats no such dietary influence was apparent. In experiment 3, strain differences in biosynthesis of α‐2u‐globulin (α2u‐g) were assessed because both α2u‐g in the urine and administration of sodium salts of organic acids such as SA have been reported to be involved in tumor promotion. Immunohistochemical analysis of renal tubules and Western blotting analysis of urine revealed the presence of α2u‐g in all three strains examined. These data suggest that differences in susceptibility to promotion are due to genetic factors rather than dietary factors and the ability to synthesize . α2u‐g.

In the two‐stage rat bladder carcinogenesis model using N‐butyl‐N‐(4‐hydroxybutyl)nitrosamine (BBN) as an initiator and sodium l‐ascorbate (SA) as a promoter, we found a notable strain difference between F344/DuCrj (F344) and WS/Shi (WS) rats in susceptibility to the promoting effect of SA. Twenty each of F344, WS and reciprocal F1hybrid rats were given 0.05% BBN in their drinking water for 4 weeks and then a basal diet with (BBN‐SA group) or without (BBN group) a 5% SA supplement for 32 weeks. In F344 and also in reciprocal F1hybrids, the number of tumors per rat was significantly higher in the BBN‐SA group than in the BBN group (P<0.0001). In contrast, WS rats were not significantly affected by either treatment (P=0.8). These findings indicate that F344 rats are highly susceptible to the promoter effect of SA, but WS rats are not. Linkage analysis of 108 WS X (WSXF344) F1backcrosses revealed that this difference was related to a quantitative trait locus mapped on rat Chr. 17 (maximum LOD score, 3.86) named Bladder Tumor Susceptible‐Iand possibly another locus on Chr. 5 (maximum LOD score, 2.39). This study has provided the first evidence that host genes influence the risk of bladder cancer development.

• A photosensitive lichen planus-like eruption developed in a 63-year-old man during postoperative chemotherapy with 1-(2-tetrahydrofuryl)-5-fluorouracil. The action spectrum for the photosensitivity was in the long-wave ultraviolet light (UV-A). The reaction was reproducible on readministration of the drug and exposure to UV-A. At the time of this test, a flare-up phenomenon was observed at previously involved sites. The photopatch test was negative with the drug. Patients taking the drug who had no dermatitis were not sensitive to UV-A. These observations suggest that the photosensitivity that occurred in the patient was possibly photoallergic.(Arch Dermatol 114:1498-1500, 1978)