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To elucidate the roles played by copper-containing enzymes in the brain degeneration associated with Menkes disease, the brains of brindled mouse hemizygotes (BMs) were studied histochemically and biochemically before and after copper therapy. Light and electron microscopic histochemistry revealed that, while neuronal mitochondria in BM brains demonstrate only a weak diaminobenzidine reaction for cytochrome oxidase, these exhibit strong activity after therapy and in control mice. Biochemical assays of enzyme activity revealed only 30% of the normal level before a single subcutaneous application of 50 µg of CuCl2, whereas neuronal mitochondria of BMs surviving 8 months after the copper therapy displayed essentially no difference from the controls. Similar results were also gained for superoxide dismutase activity, although the reduction was less marked. The present findings provide direct support for decreased activities of copper-containing enzymes being responsible for the mitochondrial abnormalities and brain degeneration associated with Menkes disease. [ABSTRACT FROM AUTHOR]

The drastic initial carcinogenic changes that induce single hepatocytes and minifoci positive for GST-P (a specific biomarker of foci and nodules) identified previously in rat livers (K. Satoh, Life Sci. 2018) require elucidation. Notably, after animals were administered benzyl isothiocyanate (BITC, anti-cancer phytochemical, 0.5% by wt) in their basal diet, immunocytochemical staining of vibratome-prepared liver specimens for GST-P revealed that the canalicular networks and bile ducts of the animal livers were heavily and finely stained for GST-P even though the biomarker is a cytosolic enzyme. In addition, the mean diameter of the canaliculi was greatly enlarged. The results thus indicate that GST-P was rapidly synthesized in all hepatocytes but rapidly excreted into bile. Similar results were obtained with animals administered dietary AAF carcinogen (0.04%). The biliary excretion of GST-P was detectable not only in all hepatocytes but also within minifoci, foci and nodules. A new initiation model was therefore proposed assuming that GST-P + single hepatocytes are formed after injury to canaliculi by carcinogens to decrease the excretion of GST-P from hepatocytes. The key findings from this study and the biomarker analysis using a vibratome technique might help elucidate the 'unknowable' mechanism of cancer initiation in rat chemical carcinogenesis., Competing Interests: Declaration of competing interest The authors declare that there are no competing interests., (Copyright © 2023. Published by Elsevier Inc.)

An improved staining method for gamma-glutamyltranspeptidase (GGT) was developed using Vibratome-prepared microslices. Microscopic precursor cell populations of preneoplastic foci positive for the marker enzyme were detectable sequentially in rat liver by tracing back from 5 to 1 week after carcinogen injection in a hepatocarcinogenesis model. Mirror-image comparisons of serial sections stained for GGT activity and immunocytochemically stained for GST-P (glutathione S-transferase P-form) revealed that GGT expression was confined within GST-P(+) cell populations (GST-P(+) minifoci), which are induced in the periportal area (zone 1) of the liver. GGT expression level differed from one minifocus to another, and the larger the GST-P(+) focus, the stronger was the GGT expression in it, indicating that GST-P(+)/GGT(-) phenotypes are convertible into proliferating GST-P(+)/GGT(+) ones. Our results suggest that there are at least 2 closely related precursors, GST-P(+)/GGT(-) and GST-P(+)/GGT(+) phenotypes, of preneoplastic foci in rat chemical hepatocarcinogenesis., ((c) 2005 Wiley-Liss, Inc.)

'De novo' carcinogenesis has been advocated besides 'adenoma carcinoma sequence' as another dominant pathway leading to the colorectal carcinoma. Our previous study demonstrated that brain (fetal)-type glycogen phosphorylase (BGP) positive foci in the transitional mucosa (BGP foci) have frequent p53 mutations and that the distribution of BGP foci has a close relationship with the location of 'de novo' carcinoma. The aims of the present study were to investigate further genetic alterations in the BGP foci and to clarify the mechanism of 'de novo' carcinogenesis. Twenty-eight colorectal carcinomas with invasion into submucosa or superficial muscularis propria without any adenoma component expressing immunoreactive p53 protein were selected from 168 resected specimens. Investigations of the p53, K-ras and APC mutations was performed in the BGP foci, BGP negative colorectal mucosa and 'de novo' carcinoma using PCR-SSCP and DNA squencing. In all 28 cases, immunoreactive BGP was positive in the carcinomas and the BGP foci were observed sporadically in the mucosa adjacent to the carcinoma. No K-ras mutation was observed in either carcinoma or BGP foci in any of the cases. Mutations of p53 and APC were 14 (50.0%) and 9 (32.1%) in 'de novo' carcinomas, and 11 (39.3%) and 1 (3.6%) in BGP foci, respectively. Both p53 and APC mutations were detected in 8 and 1, p53 mutation alone in 6 and 10, APC mutation alone in 1 and 0 out of 28 carcinomas and BGP positive foci, respectively. These results suggest that the BGP foci may play a very important role in the 'de novo' colorectal carcinogenesis from the frequent genetic alterations of p53, and that there may be two major pathways, i.e., the p53-APC pathway and the p53 alone pathway, from the chain of genetic alterations between BGP foci and 'de novo' carcinoma.

The molecular mechanism of the specific expression of glutathione S-transferase P-form (GST-P) in the rat hepatic preneoplastic foci and "GST-P-positive" single cells requires elucidation. Immunochemical and stereological analyses revealed that the enzyme level in preneoplastic foci was 150-250-fold (6.7 +/- 2.4 mg/g liver and 0.29 +/- 0.1 mM subunits) higher than in normal cells. GST-P content in the single cells was higher than in preneoplastic foci, as determined by densitometry. In addition, the single cells were larger in cell diameter and area, corresponding to 2-3-fold increase in cell volume, relative to normal cells, but showed a significant shrinkage of their nuclei. Prior to the induction of single cells in the liver by diethylnitrosamine (DEN), microsomes were severely damaged as reflected by the low yield (approximately 60% that of untreated controls) after 2 h of DEN injection. Considering that GST-P is mainly a binding protein for GSH conjugates of endogenous carcinogens, together with our findings of morphological expansion, low viability of single cells and microsomal damage, our results suggest anomalous elevation of the ligand counterparts to lethal levels in preneoplastic cells, especially in single cells. We propose that the epigenetic mechanism rather than the genetic mechanism could account for GST-P induction in hepatocytes.

The Pi-class glutathione S-transferases (GSTs) play pivotal roles in the detoxification of xenobiotics, carcinogenesis and drug resistance. The mechanisms of regulation of these genes during drug induction and carcinogenesis are yet to be elucidated. Recently, Nrf2 (NF-E2-related factor 2; a bZip-type transcription factor) knockout mice were shown to display impaired induction of Pi-class GST genes by drugs. It is known that the mouse Pi-class GST gene GST-P1 is expressed predominantly in the male liver, and is regulated by androgen. To determine whether Nrf2 and the androgen receptor regulate GST-P1 directly, we analysed the molecular mechanism of activation of this gene by these factors. The promoter of the GST-P1 gene was activated markedly by Nrf2 in transient transfection analyses. Gel mobility shift assay and footprinting analyses revealed three Nrf2 binding sites: one at the proximal and two at distal elements, located at positions -59, -915 and -937 from the cap site. The fifth intron of the GST-P1 gene contains the androgen-responsive region. Multiple androgen receptor binding sites are clustered within a 500 bp region of this intron. The whole fragment contains a minimum of seven androgen receptor binding sites, which collectively display strong androgen-dependent enhancer activity. However, on division into small fragments containing two or three elements each, individual enhancer activities were dramatically decreased. This suggests that multiple elements work synergistically as a strong androgen-responsive enhancer. Our findings indicate that Nrf2 and the androgen receptor directly bind to and activate the mouse GST-P1 gene.

Whether single cells immunohistochemically positive for glutathione S-transferase P1-1 (GSTP1-1) induced in the female mouse liver by DEN (Hatayama et al., Carcinogenesis, 14, 537-538, 1993) are precursor initiated cells of preneoplastic foci, is of importance in chemical hepatocarcinogenesis. Nrf2 transactivates a wide variety of ARE (anti-oxidant response element)-mediated enzymes including GSTP1-1. Quantitative examination revealed that the basal expression of hepatic GSTP1-1 was 60% lower in Nrf2 gene knock-out female mice(-/-) than in wild type females, and that treatment with butyrated hydroxyanisole (BHA) increased by 10-fold GSTP1-1 expression in the liver of wild type female mice but not in knockout female mice(-/-). Despite the lack of Nrf2, GSTP1-1-positive single cells were detected in livers of DEN-treated female(-/-) 3 months after treatment. Subsequent BHA feeding to the positive cell-bearing females for one more week clearly showed that the single cells were detectable with females(-/-) but not with females(+/+,+/-) due to the strong induction of GSTP1-1 in the surrounding hepatocytes. The sensitivity to DEN hepatocarcinogenesis was not significantly different among genotypes. These results demonstrate that Nrf2 is regulatory in normal hepatocytes but not in the single cells positive for GSTP1-1 inducible in the female mouse liver by DEN. The transcriptional distinction observed for the DEN-transformants is suggestive of a preneoplastic character of precursor initiated cells.

Background: Bullous congenital ichthyosiform erythroderma (BCIE) shows phenotypic variability. An epidermal nevus may represent somatic mosaicism for keratin gene mutation, which produces generalized BCIE in the next generation. This fact provides evidence that a postzygotic mutation can be passed on to the next generation in BCIE. We hypothesized that the same phenomenon occurred in a family with BCIE whose phenotypes were extremely different., Observations: We studied a 19-year-old boy with severe ichthyosiform erythroderma and prominent palmoplantar hyperkeratosis with digital contracture. In contrast, the proband's mother exhibited only mild ichthyosiform skin, granular verrucous lesions, and less severe streaky palmoplantar hyperkeratosis. Mutation analysis in the proband showed a keratin K1 mutation (N187S, ie, an A-to-G transition at the second position of codon 187, resulting in an asparagine-to-serine substitution). In the mother, the same keratin gene mutation was recognized, but only faintly in the leukocyte DNA, indicating that the amount of the mutated allele in leukocyte DNA was very low compared with that from the proband., Conclusions: We speculate that the mildly affected mother showed keratin 1 gene mosaicism, and that the BCIE phenotype had been transmitted in a severe form through a mechanism that passes the keratin gene mutation to the next generation. These results suggest that mild forms of BCIE may actually represent extensive epidermal nevi/keratin gene mosaicism.

Various bacterial pathogens have been identified as mediators of apoptosis. Apoptosis reportedly shows both detrimental and beneficial effects on biological functions. We studied the role of liver apoptosis in lethal Listeria monocytogenes infection and the regulation of apoptosis by endogenous cytokines during infection. Apoptosis was observed in the spleen but not in the liver of infected mice, whereas the induction of liver necrosis was evident by rising levels of serum aminotransferases in these animals. Apoptosis was detected in the liver of L. monocytogenes-infected mice which had been treated with monoclonal antibody (mAb) against tumor necrosis factor-alpha (TNF-alpha) or interleukin-6 (IL-6), or in TNF-alpha(-/-) mice, but not in gamma- interferon (IFN-gamma)(-/-) mice or mice which had been treated with mAb against IL-4 or IL-10. Augmentation of liver apoptosis in mice treated with mAb against TNF-alpha or IL-6 or in TNF-alpha(-/-) mice correlated with the increase in bacterial numbers in the organ, while no augmentation of apoptosis was observed in the liver of IFN-gamma(-/-) mice irrespective of the marked increase in bacterial numbers in the organs, indicating that augmentation of liver apoptosis may not be merely due to the increase in bacterial growth in the organs. These results suggest that TNF-alpha and IL-6 may play an important role in protecting the liver from apoptosis in lethal L. monocytogenes infection.

Cyclooxygenase-2 (COX-2), an enzyme responsible for catalyzing the committed step in prostanoid biosynthesis, is the product of an immediate early gene capable of being upregulated by diverse stimuli. Based on the experimental evidence that oxidative stress is associated with the upregulation of COX-2, we evaluated the effect of the oxidized fatty acid metabolites on COX-2 induction in rat liver epithelial RL34 cells. Among the compounds tested, only 4-hydroxy-2-nonenal (HNE), a highly mutagenic and genotoxic aldehyde generated during oxidative stress, dramatically induced COX-2. Enhanced gene expression of COX-2 by treatment with HNE was evident as a drastic elevation of the mRNA level. We also found that intracellular glutathione status was strictly related to HNE-induced COX-2 expression. These findings suggest the presence of a signaling pathway in the cellular response mediated by locally produced lipid peroxidation products under oxidative stress., (Copyright 2000 Academic Press.)

The active-site (the H-site) hydrophobicity of five human glutathione S-transferases (GSTs) was analyzed by application of linear free energy relationships (LFERs) with a series of S-alkylated glutathione inhibitors, GS(CH2)n - 1CH3 (n = 1-14). Distinct linear reltionships were observed in the plots of log Ki (inhibition constant) vs n for the five forms, whereby the Kis varied by three to four orders of magnitude. Mean free enthalpy changes per methylene group (-Delta DeltaG degreess), a measure of H-site hydrophobicity, were in the order M1-1 (4.6 kJ/mol) > A1-1 (3. 9 kJ/mol) > A1-2 (3.8 kJ/mol) > A2-2 (2.8 kJ/mol) > P1-1 (1.6 kJ/mol). The quantitative differences may in part account for the extraordinary broad and overlapping substrate specificities of the Alpha-, Mu-, and Pi-class isoenzymes. In contrast to the Alpha and Mu classes being selective for strongly electrophilic compounds, the neoplastic P1-1 species was indicated to be selective for weakly electrophilic and water-soluble carcinogens such as acrolein and hydroxyalkenals., (Copyright 1999 Academic Press.)

To clarify which of the two genes for pi class glutathione S-transferases (GSTs) (p-1 and p-2) is dominantly expressed in mouse hepatic adenomas, the relative mRNA levels were examined by means of the reverse transcription-polymerase chain reaction (RT-PCR). Hepatic adenomas were induced in male and female B6C3F1 mice by diethylnitrosamine treatment. Northern blot analysis revealed that pi class mRNA levels were decreased in adenomas of male mice, but increased in those of females, with reference to the respective surrounding non-adenoma tissues. In contrast to the marked sex difference in surrounding tissues, pi class GST mRNA levels in adenomas were almost the same in both males and females. To evaluate p-1 and p-2 mRNA levels separately, the products of RT-PCR employing primers common for both cDNAs were digested with the endonuclease BanI (specific for p-2) and then resolved by electrophoresis. The p-1 mRNA was thus found to be dominant in adenomas of both female and male mice. The p-2 mRNA levels were increased in the lesions as compared with those in the surrounding non-adenoma tissues. Recombinant p-1 and p-2 proteins were expressed in Escherichia coli. Unlike p-1, the p-2 protein did not show any significant activity towards 1-chloro-2,4-dinitrobenzene and did not bind to S-hexylglutathione-Sepharose despite immunological cross-reactivity. The dominant pi class form in adenomas could also be identified as p-1 by its binding to S-hexylglutathione-Sepharose. Single radial immunodiffusion analyses confirmed that the p-1 protein levels were in line with the mRNA findings, i.e., 1.9+/-0.3 mg/g adenoma as compared to 6.5+/-1.2 mg/g non-adenoma tissue for males and 2.2+/-0.6 mg/g as compared to 0.7+/-0.2 mg/g for females. The results thus indicated that the change of pi class forms in adenomas is caused mainly by alteration in the p-1 level and the contribution of p-2 is minimal.

The induction of phase II detoxifying enzymes is an important defense mechanism against intake of xenobiotics. While this group of enzymes is believed to be under the transcriptional control of antioxidant response elements (AREs), this contention is experimentally unconfirmed. Since the ARE resembles the binding sequence of erythroid transcription factor NF-E2, we investigated the possibility that the phase II enzyme genes might be regulated by transcription factors that also bind to the NF-E2 sequence. The expression profiles of a number of transcription factors suggest that an Nrf2/small Maf heterodimer is the most likely candidate to fulfill this role in vivo. To directly test these questions, we disrupted the murine nrf2 gene in vivo. While the expression of phase II enzymes (e.g., glutathione S-transferase and NAD(P)H: quinone oxidoreductase) was markedly induced by a phenolic antioxidant in vivo in both wild type and heterozygous mutant mice, the induction was largely eliminated in the liver and intestine of homozygous nrf2-mutant mice. Nrf2 was found to bind to the ARE with high affinity only as a heterodimer with a small Maf protein, suggesting that Nrf2/small Maf activates gene expression directly through the ARE. These results demonstrate that Nrf2 is essential for the transcriptional induction of phase II enzymes and the presence of a coordinate transcriptional regulatory mechanism for phase II enzyme genes. The nrf2-deficient mice may prove to be a very useful model for the in vivo analysis of chemical carcinogenesis and resistance to anti-cancer drugs.

Although the three-dimensional structure of human glutathione transferase (GST) P1-1 crystallized with a GSH analogue has been reported, its structure in the non-complexed form has not been determined. Four monoclonal antibodies to GST P1-1 were produced to facilitate structural analysis. Of these, one, clone d-1 of IgG2a isotype, dose-dependently inhibited the activity of GST P1-1 but did not affect the activities of either GST A1-1 or M1-1. On immunoblotting, the antibody reacted strongly with GST P1-1 and weakly with rat GST-P and mouse GST-II, indicating cross-reactivity with Pi-class forms but preferential reactivity with GST P1-1. When GST P1-1 and the antibody were incubated in the presence of 60 microM GSH, no inhibition of activity was found, whereas 1-chloro-2,4-dinitrobenzene had no effect at concentrations up to 10 microM. The binding of GST P1-1 to antibody adsorbed to Protein A-Sepharose was also prevented by both 0.1 mM GSH and N-ethylmaleimide treatment. Trypsin digests of GST P1-1 were resolved by HPLC and a peptide that reacted with the antibody was detected by absorption experiments. N-Terminal amino acid sequencing revealed the peptide to be in the C-terminal portion of the enzyme, stretching from amino acid residues 198 to 208. A synthetic peptide of this sequence also absorbed the antibody. These results suggest that both GSH bound to the active site and N-ethylmaleimide bound to the cysteine residue repress antibody binding to the C-terminal region. Thus this antibody may be useful for examining the steric configuration of the C-terminal and other regions of GST P1-1 in the absence of GSH.

We investigated the influence of a combination of lentinan, a biological response modifier, and cis-diamminedichloroplatinum(II) (CDDP) on the growth and glutathione S-transferase (GST) content of colon 26 tumor to examine whether lentinan represses GST expression and enhances the therapeutic effects of CDDP. Female CDF1 mice inoculated subcutaneously with transplantable colon 26 adenocarcinoma cells (1 X 10(6)/mouse) received intraperitoneal administrations of lentinan, CDDP, or the two drugs in combination, on days 10, 14, 17 and 21 after the inoculation. On day 24, tumor weights (estimated from their length and width) were significantly lower in the CDDP+ lentinan group (2.7+/-1.3 g) than in the CDDP alone group (4.3+/-0.7 g, P<0.05), both values being less than in the nontreated control group (7.2+/-1.5 g). The major GST form of colon 26 tumor was identified as GST-II, the Pi class form, and a minor form as GST-III belonging to the Mu class. Both GST-II and GST-III values on day 24 were significantly decreased in the lentinan alone (0.90+/-0.29 and 0.26 +/-0.11 microg/mg protein, respectively) and lentinan + CDDP groups (0.98+/-0.22 and 0.29+/-0.07 microg/mg protein), as compared with the control levels (1.39+/-0.20 and 0.52+/-0.11 microg/mg protein). However, these values were not different between the CDDP alone and lentinan + CDDP groups. Neither tissue interleukin (IL)-6, glutathione nor platinum values were different between the two groups. IL-6 values were elevated in about half of the samples treated with lentinan or CDDP and exhibited a modest inverse correlation with GST-II levels (r= -0.46). A GST inhibitor, ethacrynic acid, enhanced the sensitivity of cultured colon 26 cells to CDDP, suggesting the possible involvement of GST in modulating the cytotoxicity of CDDP to this cell line. These results indicated that lentinan administration decreases tissue GST-II and GST-III contents and enhances the sensitivity of colon 26 tumor to CDDP.

Since the expression of glutathione S-transferase P-form (GST-P) has been suggested from in vitro studies to be partly regulated by the oncogene product, c-Jun and c-Fos, their distributions were compared in normal rat tissues and preneoplastic hepatic lesions induced by the Solt-Farber protocol. Immunohistochemically demonstrated GST-P protein was positively correlated with expression of both c-Jun and c-Fos in the epidermis of the skin and the smooth muscle of adult lung and with either c-Jun or c-Fos respectively in the bile ducts and bronchial epithelium. However, GST-P expression was also observed in proximal and distal straight segments of the kidney and other tissues negative for c-Jun and c-Fos and both c-Jun and c-Fos were present in the renal proximal and distal convoluted tubules, where GST-P was lacking. Thus, the localization of GST-P was in some cases clearly separable from those of c-Jun or c-Fos. GST-P was found to be focally expressed from an early stage of hepatocarcinogenesis, when c-Jun was not detectable. At later stages, this oncogene product was stained in 35.7% of GST-P-positive foci, with a clear relation to the degree of GST-P staining. Since GST-P is not always accompanied by appreciable c-Jun or c-Fos, these oncogene products are apparently not prerequisites for its expression. However, c-Jun may be partly responsible for maintaining high levels of GST-P in hepatic foci at later stages of hepatocarcinogenesis.

Expression of the oncogene product c-Jun was examined and compared with that of class pi glutathione S-transferase (GST-II) during chemical hepatocarcinogenesis in female and male mice. A single i.p. injection of diethylnitrosamine (DEN) (10 mg/kg) was administered to B6C3F1 mice and livers were immunohistochemically investigated with anti-c-Jun and anti-GST-II antibodies at various time points thereafter. In females, almost all foci detected by hematoxylin and eosin staining were positive for c-Jun 3, 6, 9 and 11 months after the DEN administration. Seventy-one and 82% of c-Jun-positive foci at 9 and 11 months respectively, were also positive for GST-II, while this was the case for only 15% at 6 months. In males almost all foci were also positive for c-Jun at 3 and 6 months, but 23% of foci were negative at 9 months. Unlike the foci in females, 96 and 79% of those in males expressing c-Jun were negative for GST-II at 6 and 9 months respectively. Both GST-II expression in foci of females and its lack in those of males were highly correlated with c-Jun expression. Furthermore, single cells expressing c-Jun were also observed in both sexes at 3 months and thereafter, but not at 2 or 4 weeks. Alterations in the numbers of c-Jun-positive single cells, minifoci and foci followed sequentially revealed the number of such single cells to decrease, while foci increased, the sum being relatively constant. On the other hand, while a large number of GST-II-positive single cells were detected in female livers 2 and 4 weeks after the DEN administration, they markedly decreased thereafter, suggesting that the majority were unlikely to give rise to foci. Thus, c-Jun may be a better positive marker not only for preneoplastic foci, but also putative precursor single cells in both female and male mice and therefore be useful for analysis of hepatocarcinogenic processes.

A study has been undertaken to investigate Prostaglandin E1 administration procedure for improving flap survival. Whether the drug was administered continuously or transcutaneously using a silicone gel drug delivery system; or was topically injected into the critical zone of the flap; or was intraperitoneally administered intermittently over an hour after surgery a statistically significant improvement of flap survival occurred (P < 0.01, Student's t-test). However, no improvement of flap survival was seen when the drug was administered only once intraperitoneally immediately after flap elevation, although administered doses of the drug in those rats was equal to the doses in the rats which received intermittent administration of the drug intraperitoneally over an hour after surgery.

From a drug delivery system using silicone gel, the amount of Ofloxacin (OFLX) released or transferred to a wound and blood was measured over 2 weeks. From three types of silicone gel containing 2, 0.2 and 0.02% OFLX respectively, levels from that with 2% OFLX were highest, approximately two to five times higher than that with 0.02% OFLX. Statistically significant differences were found between the three types (P < 0.01, Student's t-test). When used in partial thickness skin wounds on rats, only an extremely small amount of OFLX was detected in the serum, being higher under gel containing 2% OFLX. In a clinical study, however, no drug was detected either in the blood or the wound after 1 week.

Using 60 Hirosaki hairless rats, the relationship between the time of starting topical administration of Prostaglandin E1 using silicone gel and flap survival rate is described. Compared to control groups, beginning the administration of PGE1 at 0 (P < 0.01) and 3 h (P < 0.05) after flap elevation resulted in an increased flap survival area (Student's t-test). However, in groups in which PGE1 administration was begun at 6, 9 and 12 h after flap elevation, no statistically significant differences could be detected.

While glutathione S-transferase P form (GST-P), a reliable marker for preneoplastic lesions induced by mutagenic hepatocarcinogens, is generally not expressed in rat liver foci, hyperplastic nodules and hepatomas induced by peroxisome proliferators (PPs), such lesions can be detected due to their peroxisomal enzyme-negative nature. For comparative purposes we examined the inducibility of enoyl CoA hydratase (ECH), a key peroxisomal enzyme, in rat hepatic preneoplastic lesions induced by mutagenic carcinogens. Clofibrate (CF) was therefore administered for 2 or 4 weeks following performance of the Solt-Farber protocol using diethylnitrosamine and 2-acetylaminofluorene. Immunohistochemical examination revealed no or only very weak expression of ECH within the induced foci in clear contrast to the strong staining of surrounding parenchyma. ECH expression was thus diametrically opposed to that of GST-P which was found only in foci. Although ECH was completely lacking in GST-P-strongly positive foci, it was expressed in GST-P-negative hepatocytes inside some foci otherwise positive for GST-P. CF administration resulted in a significant decrease in the numbers and areas of foci exhibiting strongly positive or positive GST-P staining; this being reflected in a lowering of GST-P protein levels. Furthermore, in primary cultured rat hepatocytes, clofibric acid as well as dexamethasone suppressed the expression of both GST-P and the oncogene, c-jun. These results taken together suggest that possible interaction of the PP receptor with JUN might be involved in loss of ECH expression in GST-P-strongly positive foci.

Expression of class pi glutathione S-transferase (GST-II) was investigated during chemical hepatocarcinogenesis in female and male mice. Diethylnitrosamine (DEN; 10 mg/kg body wt) was administered to B6C3F1 mice on day 15 after birth, and liver sections were immunohistochemically stained with GST-II antibody at 12 and 24 weeks thereafter. In the normal mouse liver, GST-II was strongly expressed in males but only weakly in females. At 24 weeks after DEN treatment, the majority of preneoplastic foci could be detected as GST-II-positive in the female mice, while those in males were observed as to have decreased GST-II relative to the background. Class alpha GST-I and class mu GST-III did not exhibit any remarkable changes in preneoplastic foci. This result indicates that GST-II is a useful positive and negative marker for preneoplastic lesions in female and male mouse livers. In addition, class pi GST-II may be useful for the rapid screening hepatocarcinogens in mice because GST-II-positive or -negative single cells and minifoci, considered as precursor initiated populations, were detectable in females and males at 12 weeks after the DEN injection.

To evaluate the beneficial effects of heparin on flap survival area, an experimental study was carried out on dorsal flaps in rats. Topical applications of heparin, released from a silicone gel sheet onto the critical area (which generally necrosed without treatment), resulted in an increase in the flap survival area and rate as compared to controls (p < 0.01). When heparin was applied topically but solely to an area proximal to the critical area however, no increase in the survival area resulted.

The detection of preneoplastic cells is very important for the analysis of carcinogenic processes and for developing strategies for prevention and treatment of cancer. We have been investigating enzyme alterations occurring during rat chemical hepatocarcinogenesis, especially to find more specific enzyme markers for preneoplastic hepatic lesions. We identified the placental form of glutathione S-transferase (GST-P; GST 7-7) as a new marker enzyme for preneoplastic hepatocytes. We also found human placental form, GST-pi, to be a possible tumor marker for various human tissues except liver. In this article, their properties and possible functions are reviewed on basis of our recent investigations. A peroxisomal enzyme, enoyl CoA hydratase, in also described as a possible negative marker for rat preneoplastic hepatic foci/nodules and hepatomas induced by peroxisome proliferators.

Using a silicone gel sheet continuous drug delivery system containing Prostaglandin E1 (PGE1), the relationship between flap survival rate and the site of drug application was studied. Skin flaps measuring 2 x 9 cm were raised on the dorsum of rats, and divided into 3 areas. Area 1 was within 3 cm of the flap tip, area 2 was between 3-6 cm from the flap tip including the critical zone, and area 3 was the portion within 4 cm of the flap base. Further, area 4 was a strip of skin around the flap, 1 cm in width. Compared to the control group, a significant increase in flap survival rate was seen only when PGE1 was administered to area 2 (p < 0.01, t-test). Specimens injected with dye intravascularly showed an increase in the number of thick vessels in area 3, when PGE1 was applied only to area 3. However, when PGE1 was applied to area 2, there was no significant vascular increase in area 3. Instead, an extensive network of fine vessels was observed in area 2.

Immunohistochemical staining of enoyl CoA hydratase (ECH), a key peroxisomal enzyme, revealed that the putative preneoplastic lesions induced in livers by administration of the peroxisome proliferator (PP) clofibrate (0.3% in diet) to rats for 60 weeks or more, lacked this enzyme so that they could be detected as ECH-negative foci. ECH and other peroxisomal enzymes such as acyl CoA oxidase, catalase and carnitine-dependent acetyltransferase were also either not or only weakly expressed in most hepatic hyperplastic nodules and hepatomas induced by ciprofibrate (0.025% in diet), Wy-14,643 (0.1%) or BR-931 (0.2%), while being strongly induced in surrounding hepatocytes. These results indicate that the expression of ECH and other peroxisomal enzymes is repressed in putative preneoplastic and neoplastic lesions induced by PPs in rat livers and that these peroxisomal enzymes might therefore be used as negative markers.

The mRNA levels of three phosphoseryl/phosphothreonyl protein phosphatases, PP1, PP2A and PP2C, in rat liver have been determined by Northern blot analysis in various stages of rat chemical hepatocarcinogenesis using a Solt-Farber model. Five weeks after administration of diethylnitrosamine, the mRNA levels of PP1 alpha, PP2A and PP2C were elevated 8, 29 and 11 times, respectively, as compared to those of the control livers. However, in primary hepatoma induced according to the Solt-Farber model, the mRNA levels of all three protein phosphatases were dramatically decreased to normal levels or even to much lower levels, whereas the mRNA level of glutathione S-transferase placental form, a tumor marker protein, was greatly elevated as compared with that of the control livers. In a poorly differentiated hepatoma AH13, a line of rat ascites hepatoma, the mRNA level of PP1 alpha was 5.6 times higher than that of the control livers, whereas the mRNA lever of PP2C was almost the same as that of the control livers and the level of PP2A mRNA was distinctly lower than that of the control livers. These data appear to suggest some involvement of protein phosphatases in hepatocarcinogenesis.

To clarify the role(s) of thiol (sulfhydryl) groups of cysteine (Cys) residues in the activity of the rat glutathione transferase P (7-7) form (GST-P), a cDNA clone, pGP5, containing the entire coding sequence of GST-P (Y. Sugioka et al., (1985) Nucleic Acids Res. 13, 6044-6057) was inserted into the expression vector pKK233-2 and the recombinant GST-P (rGST-P) expressed in E. coli JM109. All four Cys residues in rGST-P were independently substituted with alanine (Ala) by site-directed mutagenesis, the resultant mutants as well as the rGST-P being identical to GST-P purified from liver preneoplastic nodules with regard to molecular weight and immunochemical staining. Since all mutants proved as enzymatically active towards 1-chloro-2,4-dinitrobenzene as liver GST-P, it was indicated that none of the four Cys residues is essential for GST-P activity. However, the mutant with Ala at the 47th position from the N-terminus (Ala47) became resistant to irreversible inactivation by 0.1 mM N-ethylmaleimide (NEM), whereas the other three mutants remained as sensitive as the nonmutant type (rGST-P). Ala47 was also resistant to inactivation by the physiological disulfides, cystamine or cystine, which cause mixed disulfide and/or intra- or inter-subunit disulfide bond formation. These results suggest that the 47-Cys residue of GST-P may be located near the glutathione binding site, and modulation of this residue by thiol/disulfide exchange may play an important role in regulation of activity.

The effects of epidermal growth factor (EGF) (10 ng/ml) and insulin (100 nM) on the expression of glutathione S-transferases (GSTs), especially the GST-P form (GST 7-7), were examined in primary cultured rat hepatocytes in serum-free medium. On culture with EGF and insulin, the GST activities towards 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene were transiently decreased on day 2 to 10% of those of freshly isolated hepatocytes and then increased to 60 to 100% of those of freshly isolated cells on day 4. Western blot analysis of GSTs revealed that GST-P, which is not present in freshly isolated hepatocytes, was markedly induced and that GST subunits 3 and 4 of the Mu class also increased after addition of EGF and/or insulin, while the subunits 1 and 2 of the Alpha class disappeared. Northern blot analysis showed that on addition of EGF and insulin the level of GST-P mRNA was also elevated and expressions of the nuclear oncogenes c-jun and c-fos were enhanced. These results suggest that the enhanced expression of GST-P induced by EGF or insulin in primary cultured rat hepatocytes might be regulated by JUN and FOS proteins.

Glutathione transferases (GSTs) in Class Pi (rat GST-P (7-7) and human GST-pi) were inactivated by treatment with 0.05-1 mM hydrogen peroxide (H2O2), while GSTs in Class Alpha (1-2) and Class Mu (3-3, 3-4) were not, even with 5 mM H2O2. In the presence of 1 mM reduced glutathione (GSH), the inactivated GST-P (-pi) was effectively reactivated by the action of thioltransferase, which had been partially purified from rat liver by GSH-Sepharose affinity chromatography and gel filtration using Sephadex G-75. Thus, inactivation of GST-P by H2O2 was indicated to involve concomitant formation of disulfide bonds between cysteinyl residues. Single GST-P or GST-pi subunits are known to have four cysteinyl residues at the same positions, which can react with sulfhydryl group modifiers. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, GST-P treated with 1 mM H2O2 showed several extra bands, at least three, with apparent molecular weights of 21.5, 18, 37 kDa in addition to the native GST-P subunit band with a molecular weight of 23.5 kDa. These extra bands were identified as inactive forms since they returned to the native band with accompanying restoration of the activity when treated with dithiothreitol, mercaptoethanol, or thioltransferase. Disulfide bonds were formed mainly within subunits, causing an apparent reduction in molecular weight, only small amounts of binding between subunits being observed.

Investigation of biochemical characteristics of the glutathione S-transferase P-form (GST 7-7), a specific marker enzyme for preneoplastic cells arising during chemical hepatocarcinogenesis in the rat, revealed distinct functional differential from six other major GST forms. While the GST 7-7 substrate specificity was generally broader, binding ability for diverse organic anions such as bilirubin, hematin, and sulfobromophthalein was as high as in any of the other six forms. Furthermore, the enzymatic activity of GST 7-7 was found to be highly insensitive to the inhibitory actions of a wide range of organic anions at physiological pH in contrast to the other forms which proved more susceptible. The functional characteristics of GST 7-7 may in part account for its overproduction in the preneoplastic cells.

The activities of rat glutathione transferases (GSTs) 3-3, 3-4, 4-4 in Class mu towards 1-chloro-2,4-dinitrobenzene (CDNB) but not 1,2-dichloro-4-nitrobenzene were increased up to 5-fold during preincubation with 0.4 mM xanthine and xanthine oxidase in 50 mM potassium phosphate, pH 7.8, containing 0.1 mM EDTA. The activated GST 3-4, purified by S-hexylglutathione affinity chromatography after the treatment, had a higher specific activity (130 units/mg) than that of the nontreated (35 units/mg), the Km and Vmax values for glutathione or CDNB also were increased. Other rat GSTs in Class alpha and pi were inactivated by the same treatment. In the presence of superoxide dismutase, the activation of GST 3-4 did not occur.

Treatment of Class Pi glutathione S-transferases (GST) such as rat GST P (7-7), human GST pi and mouse GST MII with 0.05-0.1 mM N-ethylmaleimide (NEM) in 0.1 M Tris-HCl (pH 7.8) resulted in almost complete inactivation of these forms, whereas no or less inactivation occurred for GSTs in Class Alpha and Mu under the same conditions. Inactivated GST P lost its S-hexyl-GSH-Sepharose column affinity. About 0.8 mol of [14C]NEM was found to be covalently bound to 1 mol of GST P subunit when 80% of the activity was lost. Similar treatment with N-dimethyl-amino-3,5-dinitrophenyl maleimide, a colored analogue of NEM, followed by trypsin digestion, HPLC and amino acid sequence analysis revealed that one cysteine residue at the 47th position from the N-terminal of the GST P subunit was preferentially modified. Subunits of GST P and GST pi are known to have 4 cysteine residues at the same corresponding positions. The present results suggest that the 47th cysteine residue may be located in the vicinity of the active site of Class Pi GSTs.

Silicone gel sheets containing Ofloxacin (OFLX), that provide a continual drug delivery system from a wound dressing to the wound so as to prevent infection and to promote healing, are described. It was found that silicone gel sheets without added medication did not inhibit microbial growth but that gel sheets containing 0.02% and 0.2% of OFLX had a positive antimicrobial effect against Staphylococcus aureus and Pseudomonas aeruginosa in a dose-dependent fashion in vitro. Further, this antimicrobial efficacy was greatly increased in a silicone gel sheet that contained 0.02% of OFLX and an additional 10% of silicone oil. In animal experiments, a silicone gel sheet containing OFLX prevented microbiol growth and promoted rapid epithelialisation in wounds to which Staphylococcus aureus had been applied, whereas wounds covered only with OpSite all resulted in continued infection.

A new procedure using a silicone gel sheet that enables the continuous topical application of a drug to a wound, and the effect of this therapeutic procedure on experimental skin flaps of rats, are described. Although a silicone gel sheet without a drug did not augment the flap survival length, silicone gel sheets containing Prostaglandin E1 (PGE1) and Ofloxacin showed significant elongation of the survival length of the flaps without signs of infection. Similarly, silicone gel sheets containing PGE1 inserted under the flap, like a drain, also showed statistically significant augmentation of the flap survival length. These facts showed a therapeutic potential of this new silicone gel sheet containing a drug.

The fluorophotometric substrate, 7-glycidoxycoumarin (GOC), was examined for the assay of epoxide-glutathione (GSH)-conjugating activities of seven major GSH transferases (GSTs) isolated from rat liver cytosols. GST 7-7 (GST-P), isolated from the liver cytosol of rats bearing hepatic hyperplastic nodules, catalysed the GSH conjugation of GOC at a higher rate than any other examined GST isolated from the normal rat liver cytosol. GSTs 3-3, 3-4 and 4-4 (group 3-4 enzymes) had specific activities towards GOC by one fifth to one third of that of GST 7-7. GSTs 1-1, 1-2 and 2-2 (group 1-2 enzymes) had very low activities towards this epoxide. A kinetic study indicated that GST 7-7 showed the largest kappa cat/Km value for the catalytic reaction of GOC-GSH conjugation among the GSTs. In spite of their much smaller kappa cat values, group 3-4 enzymes showed much larger kappa cat/Km values for GOC than the group 1-2 enzymes, because GOC had a much higher affinity for group 3-4 enzymes than for group 1-2 enzymes. A comparative study was also done with GSH conjugations of styrene 7,8-oxide (STO) and 1-chloro-2,4-dinitrobenzene by the GSTs. Unlike GOC, the conjugation of STO was mediated at rates about twice as high by group 3-4 enzymes than by GST 7-7. STO was also a very poor substrate for group 1-2 enzymes.

When rats are subjected to chemical hepatocarcinogenesis according to the protocol of D. Solt and E. Farber ((1976) Nature (London) 263, 701-703), the liver exhibits elevated levels of tyrosine protein kinase activity as early as 3 weeks after the injection of diethylnitrosoamine. A more striking elevation in tyrosine protein kinase activity is noted in rat hepatomas induced by administration of chemical carcinogens, in particular that of 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB). Tyrosine protein kinase solubilized from the particulate fraction of 3'-Me-DAB-induced hepatoma has a molecular weight identical to that of p60v-src, cross-reacts with p60v-src immunologically, phosphorylates the heavy chain of anti-p60v-src IgG, and probably belongs to a family of p60c-src. The tyrosine protein kinase from the particulate fraction of normal rat liver is indistinguishable from the hepatoma kinase in these properties; thus it apparently differs only in the level of activity. Whether the liver and hepatoma kinases differ merely quantitatively or whether they differ even qualitatively, however, remains to be elucidated.