Depurinating naphthalene–DNA adducts in mouse skin related to cancer initiation

Naphthalene has been shown to be a weak carcinogen in rats. To investigate its mechanism of metabolic activation and cancer initiation, mice were topically treated with naphthalene or one of its metabolites, 1-naphthol, 1,2-dihydrodiolnaphthalene (1,2-DDN), 1,2-dihydroxynaphthalene (1,2-DHN), and 1,2-naphthoquinone (1,2-NQ). After 4 h, the mice were sacrificed, the treated skin was excised, and the depurinating and stable DNA adducts were analyzed. The depurinating adducts were identified and quantified by ultraperformance liquid chromatography/tandem mass spectrometry, whereas the stable adducts were quantified by ³²P-postlabeling. For comparison, the stable adducts formed when a mixture of the four deoxyribonucleoside monophosphates was treated with 1,2-NQ or enzyme-activated naphthalene were also analyzed. The depurinating adducts 1,2-DHN-1-N3Ade and 1,2-DHN-1-N7Gua arise from reaction of 1,2-NQ with DNA. Similarly, the major stable adducts appear to derive from the 1,2-NQ. The depurinating DNA adducts are, in general, the most abundant. Therefore, naphthalene undergoes metabolic activation to the electrophilic ortho-quinone, 1,2-NQ, which reacts with DNA to form depurinating adducts. This is the same mechanism as other weak carcinogens, such as the natural and synthetic estrogens, and benzene.     

Sea snake skin: Permeable to water but not to sodium

Summary Fasting yellow-bellied sea snakes (Pelamis) have a very low rate of exchange of Na with sea water. Influx and efflux are balanced at a value near 8 moles/100 g h. This is only a fraction of the rate of exchange found in marine fish. Na influx is due to uptake in the head region; dermal and cloacal influx are minimal. The impermeability of the skin to Na has been confirmed in isolated preparations. The outer keratin layer seems to be the primary barrier, since the shed skin alone is also impermeable. Na efflux can be increased to 140 m/100 g h by salt injections, and secretion by the sublingual salt gland can account for all of this loss. Fasting snakes are not in water balance in sea water. There is a net loss of water amounting to about 0.4% body wt/day that probably occurs mainly through the skin. The major osmotic problem ofPelamis in sea water seems to be water balance, not salt balance. Differences in salt gland size among sea snakes might be related to differences in skin permeability to water associated with dermal respiration. The importance of the skin as a permeability barrier suggests that the frequent skin shedding of sea snakes may be related to maintenance of low water permeability as well as to prevention of growth by marine fouling organisms.     

Cell proliferation is not required for the initiation of early cleft formation in mouse embryonic submandibular epithelium in vitro

An X-ray irradiation method was employed to analyse the role of cell proliferation in vitro in the cleft formation of mouse embryonic submandibular epithelium at early stages. When the mid 12-day gland was exposed to 200 rad of X-rays, the growth was severely retarded. In contrast, late 12-day and early 13-day glands grew apparently in a normal fashion, as did the control gland, for up to 40 h. In either case, they formed shallow clefts within 10 h of culture. With 1000 rad irradiation, the mid 12-day gland did not grow at all, but formed clefts within 20 h of culture followed by a rapid degeneration. Under the same conditions, the growth of the late 12-day gland, which was at the stage just before branching, was retarded until 10 h of culture, followed by a slight increase in epithelial size, but cleft formation was also observed within 6-10 h, as in the control gland. When exposed to a dose of 1000 rad of X-rays, the early 13-day and the late 12-day glands exhibited similar radiosensitivity; the initial narrow clefts in the epithelium deepened and new clefts began to form within 6-10 h of culture. [3H]thymidine incorporation studies revealed that a dose of 1000 rad reduced DNA synthesis of mid and late 12-day glands by 72 and 65%, respectively. Histological examination of X-irradiated late 12-day gland showed that mitotic figures were rarely seen in the epithelium at 6 h of culture. Aphidicolin, a specific inhibitor of DNA synthesis, could not halt the cleft formation of the late 12-day gland. In this experiment 89% of DNA synthesis was inhibited. Treatment of an X-ray irradiated late 12-day gland with aphidicolin blocked 92% of the DNA synthesis, but did not prevent cleft formation taking place. These results indicate that neither cell division nor DNA synthesis, is required for the initiation process of the cleft formation of the mouse embryonic submandibular epithelium at early morphogenetic stages in vitro.     

Involvement of epitope mimicry in potentiation but not initiation of autoimmune disease

We have examined whether the peptide (368-381) from the murine adenovirus type 1 E1B sequence, exhibiting a high degree of homology with the known pathogenic thyroglobulin (Tg) T cell epitope (2695-2706), can induce experimental autoimmune thyroiditis (EAT) in SJL/J mice. The viral peptide was a poor immunogen at the T or B cell level and did not elicit EAT either directly or by adoptive transfer assays. Surprisingly, however, the viral peptide was highly antigenic in vitro, activating a Tg2695-2706-specific T cell clone and reacting with serum IgG from mice primed with the Tg homologue. The viral peptide also induced strong recall responses in Tg2695-2706-primed lymph node cells, and subsequent adoptive transfer of these cells into naive mice led to development of highly significant EAT. These data demonstrate that nonimmunogenic viral peptides can act as agonists for preactivated autoreactive T cells and suggest that epitope mimicry may at times play a potentiating rather than a precipitating role in the pathogenesis of autoimmune disease.     

The initiation codon determines the efficiency but not the site of translation initiation in Chlamydomonas chloroplasts.

To study translation initiation in Chlamydomonas chloroplasts, we mutated the initiation codon AUG to AUU, ACG, ACC, ACU, and UUC in the chloroplast petA gene, which encodes cytochrome f of the cytochrome b6/f complex. Cytochrome f accumulated to detectable levels in all mutant strains except the one with a UUC codon, but only the mutant with an AUU codon grew well at 24 degrees C under conditions that require photosynthesis. Because no cytochrome f was detectable in the UUC mutant and because each mutant that accumulated cytochrome f did so at a different level, we concluded that any residual translation probably initiates at the mutant codon. As a further demonstration that alternative initiation sites are not used in vivo, we introduced in-frame UAA stop codons immediately downstream or upstream or in place of the initiation codon. Stop codons at or downstream of the initiation codon prevented accumulation of cytochrome f, whereas the one immediately upstream of the initiation codon had no effect on the accumulation of cytochrome f. These results suggest that an AUG codon is not required to specify the site of translation initiation in chloroplasts but that the efficiency of translation initiation depends on the identity of the initiation codon.     

Selenotrisulfide inhibits initiation by RNA polymerase II but not elongation

We previously reported that RNA polymerase II (purified from wheat germ) is inhibited by selenotrisulfides, the products of the reaction of selenite with sulfhydryl compounds [Frenkel, Walcott, and Middleton, Molecular Pharmacology 31, 112 (1987)]. We have now found that the initiation stage of the reaction is inhibited by selenotrisulfide but the elongation stage of the reaction is not. The actual start of the RNA chain is not inhibited by the selenotrisulfide, but rather the formation of the enzyme-DNA binary complex. Selenotrisulfide has a similar differential effect on initiation and elongation by RNA polymerase II from HeLa cells; in contrast, with E. coli RNA polymerase, it inhibits elongation as well.     

Proliferative response of mouse submandibular gland to androgen but not to thyroid hormone

Hormone-induced differentiation and proliferation of cells were investigated in the submandibular gland of castrated female mice, by determining the esteroprotease activity and 3H-thymidine labelling index, respectively. Injections of 5 alpha-dihydrotestosterone (4 micrograms/g body weight/day) or L-thyroxine (0.5 microgram/g body weight/day) induced a significant increase in the activity of esteroprotease, which has been shown to be localized in the convoluted tubule cells of the submandibular gland. Injections of the above-mentioned dose of 5 alpha-dihydrotestosterone for 3 days induced a 43-fold increase in the labelling index of the convoluted tubule cells, but injections of the above-mentioned dose of L-thyroxine for any duration did not induce a significant increase in the labelling index. The present result suggests that hormones which induce differentiation of cells in mouse submandibular gland do not necessarily induce cell proliferation.     

Human papillomavirus type 6b DNA required for initiation but not maintenance of transformation of C127 mouse cells.

We describe the transformation of C127 mouse fibroblasts with human papillomavirus type 6b (HPV-6b) DNA, which is associated primarily with benign tumors of the human genital tract. The major transformed phenotype of the HPV-6b-transfected cells lines, which had been G418 selected, pooled, and maintained without subsequent selection, was tumorigenicity in nude mice. We found that, unlike that reported for other HPVs or papovaviruses, the transformed phenotype was expressed after a delay, in which the cells had undergone extensive culture passages (about 20 passages or 100 generations). Interestingly, the HPV-6b DNA had become reduced or nondetectable in copy number in the cells by the time the transformed phenotype was expressed and in most of the tumors induced by the cells in nude mice, indicating that high levels of HPV-6b DNA were not required for maintenance of the transformed phenotype. Clonal cell lines gave similar results. When continued G418 selection was used to maintain high-copy-number HPV-6b DNA, the cells were tumorigenic, indicating that high levels of HPV-6b DNA did not suppress tumorigenesis. These studies suggest that HPV-6b DNA initiates transformation of C127 cells but is dispensable for expression or maintenance of the transformed phenotype. Transformation by HPV-6b DNA in vitro may provide insights into the HPV type-specific association with benign versus malignant lesions in vivo and may elucidate some of the oncogenic processes involved in tumor progression.     

Enhanced papilloma formation in response to skin tumor promotion in transgenic mice overexpressing the human ornithine decarboxylase gene.

We have studied the induction of papilloma formation in response to skin tumor promotion in transgenic mice overexpressing the human ornithine decarboxylase gene and in their nontransgenic littermates. The transgenic animals displayed a basal epidermal ornithine decarboxylase activity that was nearly 20 times higher than in their nontransgenic littermates. A single topical application of 12-O-tetradecanoylphorbol-13-acetate induced a much more profound and longer-lasting increase in transgene-derived ornithine decarboxylase activity in comparison with the endogenous enzyme activity. Initiation of skin tumorigenesis with a single topical application of dimethylbenz[a]antracene followed by twice-weekly application of 12-O-tetradecanoylphorbol-13-acetate resulted in the appearance of first papillomas both in nontransgenic and transgenic animals by week 7. However, after 11 weeks of 12-O-tetradecanoylphorbol-13-acetate application, the number of papillomas per animal was almost 100% higher in the transgenic animals than in their nontransgenic littermates. These results indicate that an overexpression of epidermal ornithine decarboxylase confers a growth advantage on skin tumors in vivo.     

Exposure of mouse oocytes to bisphenol A causes meiotic arrest but not aneuploidy

Mouse oocytes isolated from large antral follicles were exposed to a wide range of concentrations of bisphenol A (BPA) during maturation in vitro (50 ng/ml to 10 microg/ml BPA in medium). Exposure to high concentrations of BPA (10 microg/ml) affected spindle formation, distribution of pericentriolar material and chromosome alignment on the spindle (termed congression failure), and caused a significant meiotic arrest. However, BPA did not increase hyperploidy at meiosis II at any tested concentration. Some but not all meiosis I arrested oocytes had MAD2-positive foci at centromeres of chromosomes in bivalents, suggesting that they had failed to pass the spindle checkpoint control. In a second set of experiments prepubertal mice were exposed sub-chronically for 7 days to low BPA by daily oral administration, followed by in vitro maturation of the denuded oocytes to metaphase II in the absence of BPA, as this treatment protocol was previously reported to induce chromosome congression failure and therefore suspected to cause aneuploidy in oocytes. The sub-chronic exposure subtly affected spindle morphology and oocyte maturation. However, as with the exposure in vitro, there was no evidence that low BPA doses increased hyperploidy at meiosis II. In conclusion, the data suggest that mouse oocytes from mice respond to BPA-induced disturbances in spindle formation by induction of meiotic arrest. This response might result from an effective checkpoint mechanism preventing the occurrence of chromosome malsegregation and aneuploidy. Low chronic BPA exposure in vivo as such does not appear to pose a risk for induction of errors in chromosome segregation at first meiosis in mouse oocytes. Additional factors besides BPA may have caused the high rate of congression failure and the temporary increase in hyperploidy in mouse metaphase II oocytes reported previously.     

The effect of stretching on formation of myofibroblasts in mouse skin

Summary Wound contraction results from the contractile activity of modified fibroblasts, termed myofibroblasts, which are present in the granulation tissue of the healing wound. This study examines the relative role of mechanical tension (stretching) and wound healing as events capable of stimulating the formation of myofibroblasts in mouse skin. The skin of hairless mice was subjected to mechanical stretching and to a small incisional wound either separately or in combination. Animals were killed at intervals between 1 and 6 days and the dermis examined with the electron microscope. Stretching alone produced little evidence of inflammation at any time interval but cells with the ultrastructural characteristics of myofibroblasts were present at 4 days and abundant at 6 days. Skin that had been both stretched and wounded showed a marked inflammatory response and also contained myofibroblasts, but they were less frequent than in the skin subjected to stretching alone. Very few myofibroblasts were evident in skin that had only been wounded. It is suggested that the effect of mechanical tension alone may initiate formation of myofibroblasts in a tissue.     

Exposure to retinoic acid in the neonatal but not adult mouse results in synchronous spermatogenesis

Retinoic acid (RA) is required for germ cell differentiation, the regulation of which gives rise to a constant production of mature sperm. In testes from 3-day postpartum (dpp) RARE-hsplacZ mice, periodic regions positive for beta-galactosidase activity were observed along the length of the seminiferous tubules. Periodicity was abolished by treatment of neonates with exogenous RA at 2 dpp. To assess the consequences, 2-dpp mice were treated with RA, and the long- and short-term effects were assessed. Long-term effects of neonatal RA exposure included a delay in the appearance of advanced germ cells and the absence of a spermatogenic wave (synchronous spermatogenesis) in the adult. In contrast, RA exposure in vitamin A-sufficient adults did not result in synchronous spermatogenesis but rather induced apoptosis in a subset of spermatogonia. Shortly after (24 h) neonates were exposed, altered expression of known germ cell differentiation and the (Stra8, Kit, Sycp3, and Rec8) meiosis markers and an increase in the number of STRA8 and SYCP3 immunopositive cells were observed relative to those of vehicle controls. However, 48 and 72 h after exposure, a significant reduction in the number of STRA8 and SYCP3 immunopositive cells occurred. Immunohistochemical analysis of a marker for apoptosis demonstrated neonatal exposure resulted in increased germ cell apoptosis, as observed in the adult. Additionally, RA exposure resulted in increased Cyp26a1 expression of the RA-degrading enzyme. Thus, while RA treatment of neonatal and adult mice resulted in apoptosis of spermatogonia, synchronous spermatogenesis occurred only after neonatal RA exposure.     

Simulated resistance training, but not alendronate, increases cortical bone formation and suppresses sclerostin during disuse

Mechanical loading modulates the osteocyte-derived protein sclerostin, a potent inhibitor of bone formation. We hypothesized that simulated resistance training (SRT), combined with alendronate (ALEN) treatment, during hindlimb unloading (HU) would most effectively mitigate disuse-induced decrements in cortical bone geometry and formation rate (BFR). Sixty male, Sprague-Dawley rats (6-mo-old) were randomly assigned to either cage control (CC), HU, HU plus either ALEN (HU+ALEN), or SRT (HU+SRT), or combined ALEN and SRT (HU+SRT/ALEN) for 28 days. Computed tomography scans on days -1 and 28 were taken at the middiaphyseal tibia. HU+SRT and HU+SRT/ALEN rats were subjected to muscle contractions once every 3 days during HU (4 sets of 5 repetitions; 1,000 ms isometric + 1,000 ms eccentric). The HU+ALEN and HU+SRT/ALEN rats received 10 μg/kg ALEN 3 times/wk. Compared with the CC animals, HU suppressed the normal slow growth-induced increases of cortical bone mineral content, cortical bone area, and polar cross-sectional moment of inertia; however, SRT during HU restored cortical bone growth. HU suppressed middiaphyseal tibia periosteal BFR by 56% vs. CC (P < 0.05). However, SRT during HU restored BFR at both periosteal (to 2.6-fold higher than CC) and endocortical (14-fold higher than CC) surfaces (P < 0.01). ALEN attenuated the SRT-induced BFR gains during HU. The proportion of sclerostin-positive osteocytes in cortical bone was significantly higher (+121% vs. CC) in the HU group; SRT during HU effectively suppressed the higher proportion of sclerostin-positive osteocytes. In conclusion, a minimum number of high-intensity muscle contractions, performed during disuse, restores cortical BFR and suppress unloading-induced increases in sclerostin-positive osteocytes.     

Sooty foot, a novel mouse mutation that affects the pigmentation of exposed skin, but not hair, maps to Chromosome 2

We have characterized a novel recessive mouse mutation, named sooty foot, that increases the pigmentation of the exposed skin on the foot pads, the genital region, around the snout and muzzle, the ears, and the tail. By contrast, the pigmentation of the hair is unaffected. We have localized the mutation to Chromosome 2 by polymerase chain reaction (PCR) amplification of simple sequence repeats from pooled DNA from backcross progeny. In an extended backcross we have generated a detailed map of the region around sooty foot. Received: 25 February 1997 / Accepted: 26 April 1997     

Long-term high-fat feeding leads to severe insulin resistance but not diabetes in Wistar rats

Although lipid excess can impair beta-cell function in vitro, short-term high-fat feeding in normal rats produces insulin resistance but not hyperglycemia. This study examines the effect of long-term (10-mo) high polyunsaturated fat feeding on glucose tolerance in Wistar rats. The high fat-fed compared with the chow-fed group was 30% heavier and 60% fatter, with approximately doubled fasting hyperinsulinemia (P < 0.001) but only marginal fasting hyperglycemia (7.5 +/- 0.1 vs. 7.2 +/- 0.1 mmol/l, P < 0.01). Insulin sensitivity was approximately 67% lower in the high-fat group (P < 0.01). The acute insulin response to intravenous arginine was approximately double in the insulin-resistant high-fat group (P < 0.001), but that to intravenous glucose was similar in the two groups. After the intravenous glucose bolus, plasma glucose decline was slower in the high fat-fed group, confirming mild glucose intolerance. Therefore, despite severe insulin resistance, there was only a mildly elevated fasting glucose level and a relative deficiency in glucose-stimulated insulin secretion; this suggests that a genetic or congenital susceptibility to beta-cell impairment is required for overt hyperglycemia to develop in the presence of severe insulin resistance.