Molecular nature of ultraviolet B light-induced deletions in the murine epidermis.

Depletion of the stratospheric ozone layer leads to an increase in ambient UV loads, which are expected to raise skin cancer incidences. Tumor development in the skin could be a multistep process in which various genetic alterations, such as point mutations and deletions, occur successively. Here, we demonstrate that UVB irradiation efficiently induces deletions in the epidermis using a novel transgenic mouse, gpt delta. In this mouse model, deletions in lambda DNA integrated in the chromosome are preferentially selected as Spi(-) (sensitive to P2 interference) phages, which can then be subjected to molecular analysis. The mice were exposed to UVB at single doses of 0.3, 0.5, 1.0, 1.5, and 2.0 kJ/m(2). After 4 weeks, lambda phage was rescued from the genomic DNA of the epidermis by in vitro packaging reactions. The mutant frequencies of Spi(-) with large deletions in the epidermis increased >15-fold at a UVB dose of 0.5 kJ/m(2) over the control. Molecular sizes of most of the large deletions were >1000 bp. More than one-half of the large deletions occurred between short direct-repeat sequences from 1 to 6 bp, and the remainder had flush ends. In the unirradiated mouse, almost all of the Spi(-) mutants were 1-bp frameshifts in runs of identical bases. These results suggest that UVB irradiation induces deletions in the murine epidermis, and most of the deletions are generated through end-joining of double strand breaks in DNA.

Defective control of apoptosis, radiosensitivity, and spindle checkpoint in ataxia telangiectasia.

We examined the regulation of apoptosis, radiosensitivity, and spindle checkpoint in response to DNA-damaging agents in ataxia telangiectasia (AT)-derived lymphoblastoid cell lines (AT-LCLs), which lack AT mutated (ATM) protein expression. In addition to the previous findings that AT-LCLs are defective in regulation of cell cycle at the G1, S, and G2-M checkpoints in response to X-ray irradiation (X-IR) and are highly sensitive to X-IR (J. Biol. Chem., 271: 20486-20493, 1996), we showed for the first time that AT-LCLs were defective in X-IR-associated spindle checkpoint control. The cells were also resistant to early apoptosis as much as LCLs derived from patients with Li-Fraumeni syndrome (LFS-LCLs). Terminal deoxynucleotidyl transferase-mediated nick end labeling assay of LCLs, however, demonstrated a significant increase in apoptotic cells among AT-LCLs cultured over a longer period after X-IR. These findings were in contrast to those of LFS-LCL, which showed very little increase in terminal deoxynucleotidyl transferase-mediated nick end labeling-positive population, even in cells with hyperploidy. Thus, although early apoptosis and cell cycle controls in response to DNA damage are disrupted in both ATM and p53 mutations, cells from AT patients are much more susceptible to late-onset apoptosis than those of LFS. These differences may depend on the level of accumulation of DNA damage and/or threshold that triggers late-onset cell death in ATM or p53 mutations. Our findings allow a better understanding of the role of ATM in p53-dependent and independent signal transduction pathways in response to DNA damaging agents.

Defective control of apoptosis and mitotic spindle checkpoint in heterozygous carriers of ATM mutations.

Ataxia telangiectasia (AT) carrier-derived lymphoblastoid cell lines (AT-LCLs/hetero) with suboptimal ATM protein expression were examined for the regulation of radiosensitivity, apoptosis, and mitotic spindle checkpoint in response to DNA-damaging agents. Although AT-LCLs/hetero showed intermediate radiation sensitivity, as determined by clonogenic assay, they were resistant to early-onset apoptosis, as much as AT patient-derived LCLs (AT-LCLs/homo). Furthermore, two of three AT-LCLs/hetero showed defective mitotic spindle checkpoint control in response to X-ray irradiation, which is a recently characterized biological feature in AT-LCLs/homo. Our findings indicate that carriers of ATM mutation have biological abnormalities due to haploinsufficiency of ATM protein or dominant-negative effect of mutant ATM protein. Thus, although it is still controversial whether ATM mutation carriers are at higher risk for cancer during adulthood, our findings based on in vitro biological indicators support the notion that at least some of such carriers are at a higher risk for cancer development than those without ATM mutation. Our findings may help to reevaluate epidemiological studies on cancer susceptibility in AT carriers.

Effects of dietary docosahexaenoic acid on surface molecules involved in T cell proliferation.

It is known that n-3 polyunsaturated fatty acids (PUFA) such as docosahexaenoic acid (DHA) suppress immunity as compared with n-6 PUFA such as linoleic acid (LA), but the mechanism involved in this phenomenon is still unclear. The present study was designed to assess the effect of dietary DHA on the surface molecules involved in T cell proliferation. Weanling male C57BL/6 mice were divided into four dietary groups that were fed a 10% fat diet for 4 weeks varying in amounts of DHA and LA. As the dietary DHA concentration increased, the surface expression of CD4 and CD8 on splenic T cells decreased, while that of CD28 increased. The surface expression of CD3, however, was invariable in all dietary groups. DNA synthesis of splenic T cells, induced by CD3 crosslinkage with anti-CD3 epsilon monoclonal antibody in the presence of CD28-mediated costimulation, increased as the DHA concentration was elevated. These observations suggest that diets rich in DHA exert some of their immunomodulatory effects by a downregulation of surface expression of CD4 and CD8 and by an upregulation of CD28-mediated costimulatory signal.

DNA damage-associated cell cycle and cell death control is differentially modulated by caffeine in clones with p53 mutations.

Caffeine is known to potentiate the cytotoxic effects of DNA damaging agents and increases the sensitivity of p53-deficient cells to X-irradiation (X-IR). We have analyzed the cell cycle and cell death control after X-IR in the absence or presence of caffeine in hematological cell lines with various configurations of the p53 gene; EBV-immortalized lymphoblastoid cells with heterozygous p53 mutation (wt/mt), human leukemia cell lines HL60 and KOPM28 with no and mutant p53 expression, respectively. These cell lines display an impaired G0/G1 checkpoint and G2 delay following X-IR, and resistance to apoptosis, which are in accordance with findings previously reported. When irradiated in combination with caffeine, all these cell lines overrode the G2 delay and accumulated at G0/G1. The cell cycle modifications in these cell lines correlated with the increase in radiation-induced p34Cdc2 kinase activity by caffeine. These cell cycle control modifications by caffeine, however, were not associated with enhancement of radiation-induced apoptosis or reduction of clonogenic growth activity in these cell lines. These results suggest that the cytocidal effect of caffeine may need to be verified independently of its cell cycle regulatory activities at least in some cases with p53 mutation.

Oral contraceptive steroids: effects on iron and zinc levels and on tryptophan pyrrolase and alkaline phosphatase activities in tissues of iron-deficient anemic rats.

Weanling Wistar-strain female rats were fed a normal or an iron-deficient diet for 8 weeks and oral contraceptive steroids (OCS) were added for the last 4 weeks. Hemoglobin content, serum iron and zinc levels, liver iron levels, and tryptophan pyrrolase activities, and liver, kidney, and brain zinc levels and alkaline phosphatase activities were determined. Compared to control rats given the normal diet (N group), elevated liver zinc levels and tryptophan pyrrolase activity were found in rats fed the normal diet containing OCS ( + S group), but other parameters did not alter. In rats fed the iron-deficient diet alone (D group), only liver zinc levels were significantly higher, while other parameters were in general lower than those in the N group. In rats fed the iron-deficient diet containing OCS (D + S group), all hematological values, tissue mineral contents with the exception of liver zinc levels, and liver tryptophan pyrrolase and kidney alkaline phosphatase activities were lowered, compared to the N or N + S group. However, compared to the D group, the values of most parameters in the D + S group did not differ significantly, apart from an increase in serum zinc levels. These observations suggest that OCS does not greatly influence the various changes caused by iron-deficient anemia.

Mutagenicity of 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) in the new gpt delta transgenic mouse.

Gender differences and organ specificity of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced mutagenesis were examined with the new gptdelta transgenic mouse (T. Nohmi, M. Katoh, H. Suzuki, M. Matsui, M. Yamada, M. Watanabe, M. Suzuki, N. Horiya, O. Ueda, T. Shibuya, H. Ikeda, T. Sofuni, A new transgenic mouse mutagenesis test system using Spi-and 6-thioguanine selections (Environ. Mol. Mutagen. 28 (1996) 465-470). In this mouse model, two distinct selections are employed to efficiently detect different types of mutations, i.e 6-thioguanine (6-TG) selection for point mutations and Spi-selection for deletions, respectively. In both selections, the highest mutant frequencies were observed in colon, followed by in spleen and liver. No increases in mutations were observed in testis, brain and bone marrow in PhIP-treated male mice. No significant differences in 6-TG and Spi- mutant frequencies were observed in colon and liver between male and female treated mice. The correlation between PhIP-induced mutagenesis and carcinogenesis in colon is discussed.

Proteoglycan complexes from bovine heart valve. Fractionation by density-gradient centrifugation and gel filtration under dissociative conditions.

Proteoglycan complexes from collagenase [EC 3.4.24.3]-indigestible materials of bovine heart valves were extracted with 4 M guanidinium chloride, purified by ion-exchange column chromatography in a urea-containing solution, then fractionated by density-gradient centrifugation under dissociative conditions. Electrophoretic characteristics and enzymic susceptibility of the density-gradient fractions revealed that the glycosaminoglycans constituting the proteoglycan complexes in this indigestible materials were mainly dermatan sulfate in the top three fractions, and dermatan sulfate and chondroitin sulfates in the bottom fraction; a minor constituent which was common to all the fractions was hyaluronic acid. A gel-like substance (Fr. Ig) at the top of the gradient, amounting to about 25% of the loaded dry sample, contained only a trace of hydroxyproline (less than 1%) and was composed of proteodermatan sulfate, glycoprotein, and a small amount of hyaluronic acid. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analyses of Fr. Ig with 2-mercaptoethanol showed that the major part of the proteins in this gel-like substance was cross-linked by disulfide bridges. Chromatography of Fr. Ig on Sepharose 4B in buffered 4 M guanidinium chloride containing 2-mercaptoethanol, together with the electrophoretic patterns of the resulting fractions, suggested that proteodermatan sulfate was not associated with hyaluronic acid through covalent bonds. The amino acid composition of Fr. Ig was very similar to that reported in the literature for "dermatan sulfate-protein complex", and "structural glycoprotein" or "acidic structural protein".

UVB-induced gpt mutations in the skin of gpt delta transgenic mice.

Ultraviolet light B (UVB)-induced mutagenesis was studied in gpt delta transgenic mice, which contain the lambdaEG10 shuttle vector as a transgene. The mice were exposed to UVB at single doses of 0.3, 0.5, 1.0, 1.5, and 2.0 kJ/m(2). At 4 weeks after irradiation, the mutant frequencies (MF) of the gpt gene were determined in the epidermis and the dermis, and the gpt mutations in the epidermis were identified by DNA sequencing. The epidermis exhibited a higher sensitivity to UVB than the dermis at doses of 0.3 and 0.5 kJ/m(2) UVB: the MF of the epidermis were more than nine times higher than those of the nonirradiated mice, whereas the MF of the dermis were only two to three times higher than the nonirradiated level at the doses used. The UVB-induced mutation spectrum in the epidermis was dominated by G:C to A:T transitions at dipyrimidine sites, such as 5'-TC-3', 5'-CC-3', and 5'-T/C-CG-3'. Tandem transitions such as CC to TT were also observed. Interestingly, a remarkable bias towards the template strand of the gpt gene was observed in the single transitions at 5'-TC-3' and 5'-CC-3' sites, but not at 5'-T/C-CG-3' site. In contrast, G:C to A:T transitions at CpG sites and deletions were observed in nonirradiated mice. Hot spots of transitions were observed at different sites in UVB-irradiated and nonirradiated mice. These results indicate that gpt delta transgenic mouse is a suitable model for studying in vivo UVB-induced mutations at the molecular level.

Dietary docosahexaenoic acid can alter the surface expression of CD4 and CD8 on T cells in peripheral blood.

The effect of dietary docosahexaenoic acid (DHA) on T cell states in peripheral blood was investigated. Weanling male C57Bl/6N mice were kept on one of three 10% fat diets containing various amounts of DHA and linoleic acid for 4 weeks. Changing the concentration of dietary DHA did not alter the proportion of T cells expressing CD4 or CD8. However, increasing the concentration of dietary DHA lowered the expression of CD4 and CD8 on the cell surface. The decreased expression of these surface molecules involved in T cell proliferation has serious implications in the role of DHA as an immunosuppressant.

Secretion and excretion of immunoglobulin A to cecum and feces differ with type of indigestible saccharides.

The study was conducted to elucidate the effects of orally administered indigestible saccharides (IDS) on immunoresponses of the intestinal tracts, especially secretion and excretion of immunoglobulin A (IgA). Male 4-week-old Sprague-Dawley rats were fed diets containing several kinds of IDS (cellulose, corn husk, glucomannan, curdlan and lactulose) at 5% for three weeks. The results indicated that the proportion of IgA-presenting lymphocytes in the cecal mucosa of the tested IDS groups increased significantly or tended to increase compared with that of the cellulose group. No significant differences among the experimental groups were observed in the CD4(+)- and CD8(+)-presenting lymphocytes and the CD4+/CD8+ ratios in the small intestine, cecum and mesenteric lymph nodes. IgA amounts in the cecal contents increased significantly in the glucomannan and curdlan groups as compared with that in the cellulose group. The inconsistent results were observed in the cecal IgA amounts of the lactulose group. Although IgA excretion into feces increased periodically in the cellulose, hardly any changes were observed in the glucomannan and curdlan groups. These results revealed that IgA secretion from cecal mucosa to contents was promoted, and its excretion to feces was decreased by the oral administration of highly fermentable IDS, respectively, while non- or low-fermentable IDS functioned adversely to IgA responses in the intestinal tract. It is suggested that the response of IgA in the intestinal immune system differs with the type of IDS ingested.

High phosphorus diet rapidly induces nephrocalcinosis and proximal tubular injury in rats.

The development of nephrocalcinosis and the time course of changes in kidney function, especially proximal tubular function, were studied in young male rats fed a high-phosphorus diet. The animals were fed a purified diet with a phosphorus content of either 0.5% (normal phosphorus diet) or 1.5% (high-phosphorus diet). In the group fed the high-phosphorus diet, nephrocalcinosis was found in 4 of 42 rats after 1 d of feeding and in all rats of this group at 3 d. The degree of nephrocalcinosis gradually increased with time. Upon histological observation by electron microscopy, vacuoles, lysosomes and swelling of microvilli in the proximal tubules were observed in rats fed the high-phosphorus diet after 1 d of feeding. Giant lysosomes with deposition of calcium and deposition of hydroxyapatite in mitochondria were observed in the proximal tubules of rats fed the high-phosphorus diet at 3 d. Albumin concentration in the urine of these rats was significantly increased at 3 d. The activity of N-acetyl-beta-D-glucosaminidase in the urine was also significantly increased after 1 d of feeding the high-phosphorus diet, and then reached a plateau. The beta 2-microglobulin concentration in the urine of rats fed the high-phosphorus diet was significantly increased at 14 d, and increased more toward 21 d. We concluded that nephrocalcinosis and injury to the proximal tubules are rapidly induced in rats fed a high-phosphorus diet.

Effect of indigestible saccharides on B lymphocyte response of intestinal mucosa and cecal fermentation in rats.

The effects of water-soluble and -insoluble indigestible saccharides (IDS) on immune responses of the intestinal tract were studied. Male 4-week-old Sprague Dawley rats were fed for three weeks on diets containing several kinds of IDS at 5%. The results revealed that the proportion of kappa-light chain and IgA-presenting lymphocytes in small intestinal and cecal mucosa differed in increased number depending on the type of IDS. The response of colonic mucosa was not pronounced. The amounts of short-chain fatty acid (SCFA) and lactic acid in the cecal contents of the other test groups except the celfur group tended to be higher than those in the cellulose group, particularly in the lactulose group where many acids showed significant increases. The correlation between the proportion of kappa-light chain and IgA-presenting lymphocytes in the cecal mucosa and lactic acid in the cecal contents was significant, but that between the proportion of both lymphocytes and SCFA was not. Based on the above, we concluded that the oral administration of IDS induces the proliferation of kappa-light chain and IgA-producing B lymphocytes in small intestinal and cecal mucosa, but the degree of response differs depending on the type of IDS. It is thus suggested that IDS are involved in the intestinal immune system of rats.

Effect of vitamin K2 on experimental calcinosis induced by vitamin D2 in rat soft tissue.

The effect of vitamin K2 on calcium (Ca) and inorganic phosphorus (P) levels in the aorta and kidney obtained from experimental calcinosis induced by vitamin D2(2.5 x 10(5) I.U./ kg b.w.) of male rats was investigated. A high dose of vitamin K2 (100 mg/kg b.w.) inhibited the increase in the aortic Ca and P or in the renal Ca and P induced by vitamin D2, and a low dose of vitamin K2 (10 mg/kg b.w.) showed the same tendency, but the degree of the efficacy was small. It may be suggested that a high dose of vitamin K2 suppressed experimental calcification of soft tissues induced by vitamin D2. Therefore, a pharmacological dose of vitamin K2 might have a usefulness for the prevention and treatment of arteriosclerosis with calcification.

Effects of oral contraceptive steroids on aortic collagen, elastin and cholesterol levels in iron-deficient rats.

The effects of oral contraceptive steroids (OCS) and/or Fe deficiency on aortic collagen, elastin and cholesterol levels in female rats were investigated for 12 months. The Fe deficiency did not affect the aortic collagen and cholesterol levels, but it had a tendency to decrease the elastin content. The administration of OCS tended to increase the collagen content, while it did to lower the elastin levels. The combination effects of OCS treatment and the Fe deficiency on the aortic components were in general similar to those of OCS or Fe-deficient anemia alone. The data suggest that chronic treatment with OCS under the Fe deficiency may not greatly alter rat cardiovascular function by modifying the aortic collagen, elastin, and cholesterol contents.

Effects of vitamin E deficiency on hepatic microsomal cytochrome P450 and phase II enzymes in male and female rats.

The effects of a vitamin E (VE) deficient diet given for 120 days on total cytochrome P450 (P450) contents and UDP-glucuronyl transferase (UDPGT) and sulfotransferase (ST) activities were investigated in rat liver of both sexes. The VE deficient regimen lowered the serum VE levels, increasing the hepatic malondialdehyde concentration regardless of animal sex, which shows an elevation of the tissue peroxi-disability. Hepatic total P450 contents were significantly decreased in male rats given the VE deficient diet, whereas the effects on female rats were less obvious. Concerning phase II enzymes, VE deficiency had no effect on the activities of UDPGT and ST in rats of both sexes. The data suggest that overall capacity of detoxification of xenobiotics may be impaired by VE deficiency to a much greater extent in male than in female rat liver.

Effects of capsaicin and ethanol on hepatic drug-metabolizing enzymes in rat.

Capsaicin (CAP; 50 mg/kg body wt., p.o.) and/or ethanol (EtOH; 10% (v/v) in the drinking water) were given for a period of 30 days to male rats, and changes in hepatic drug-metabolizing enzymes were investigated. The EtOH alone tended to increase the microsomal parameters such as cytochrome P-450 content and enzyme activities of aniline hydroxylase, aminopyrine demethylase and UDP-glucuronyl transferase, while it did not affect the cytosolic enzyme activities of sulfotransferase and glutathione S-transferase. The administration of CAP without EtOH tended to decrease all of the parameters studied. In contrast, the ingestion of CAP with EtOH tended to increase even the elevated microsomal enzyme activities by EtOH alone. These data suggest that chronic alcohol ingestion could modify the potential for detoxification of xenobiotics in persons who regularly consume a large amount of hot peppers.

Long-term exposure of Wistar rats to high dietary sodium chloride level. I. Changes in aortic connective tissue components.

The effect of chronic salt loading on aortic components of connective tissue was evaluated in normotensive Wistar rats maintained on a high sodium chloride (NaCl; 8%) regime for a period of 30, 90, 180 and 360 days. The blood pressure remained unchanged until 90 days and was significantly elevated thereafter. The high NaCl diet for 180 days influenced neither the serum levels of both total cholesterol and triglyceride nor the aortic contents of collagen, elastin and glycosaminoglycans (GAG) derived from the proteoglycans. The only significant change associated with a 360-day massive NaCl ingestion was the increased aortic concentration of total GAG due to an increase in chondroitin sulfate, dermatan sulfate and heparan sulfate rather than hyaluronic acid. This study suggests that an increase in the contents of sulfated GAG occurred in the late stage of hypertensive aorta of NaCl loaded rats might be responsible, at least in part, for the pathogenesis of atherosclerotic cardiovascular disease.

Long-term exposure of Wistar rats to high dietary sodium chloride level. II. Changes in hepatic drug-metabolizing and glutathione-related enzyme systems.

The effect of excess sodium chloride (NaCl) intake on hepatic drug-metabolizing and glutathione-related enzyme systems in rats was investigated for a period of 360 days. There was no change in any parameters studied until day 180 of the experiment. However, feeding of a 8% NaCl diet for 360 days produced a decrease in cytochrome P-450 content and an increase in glutathione peroxidase activity, and resulted in a lowered capacity of hepatic tissue to activate benzo[a]pyrene to mutagens detectable in the Ames bacterial mutagenesis assay. These observations suggest that although there may be little possibility of the involvement of high NaCl diet in the carcinogenic effects of benzo[a]pyrene, long-term excess NaCl intake could reduce the potential for detoxification of many xenobiotic compounds leading to various pathological process.