Modulatory effect of glucose-6-phosphate dehydrogenase deficiency on benzo(a)pyrene toxicity and transforming activity for in vitro-cultured human skin fibroblasts.

Human skin fibroblasts isolated in vitro from subjects carrying the Mediterranean variant of glucose-6-phosphate dehydrogenase exhibit an 85% decrease of this enzymatic activity. There is a 26% and a 94% decrease of the hexose monophosphate shunt and of the reduced nicotinamide adenine dinucleotide phosphate/nicotinamide adenine dinucleotide phosphate ratio, respectively. Incubation with 0.1 mM methylene blue activates the hexose monophosphate shunt 7 times that of normal fibroblasts and only 2.2 times that of glucose 6-phosphate-deficient cells. This behavior is coupled with an increase of the resistance to cell death induced by benzo(a)pyrene, a carcinogen, the activation of which proceeds through a reduced nicotinamide adenine dinucleotide phosphate-dependent arene oxide formation. In contrast, no difference between the normal and the deficient fibroblasts exists as regards the toxic effect of methylnitrosourea, a carcinogen that does not need metabolic activation. A growth-retarding effect of benzo(a)pyrene was observed in both normal and deficient cells during 9 days in vitro. This effect is lower in the fibroblasts carrying the Mediterranean glucose-6-phosphate dehydrogenase variant. Glucose-6-phosphate dehydrogenase deficiency protects human fibroblasts against the benzo(a)pyrene-induced in vitro transformation. This effect is mimicked by the incubation of normal fibroblasts with dehydroepiandrosterone, a strong inhibitor of glucose-6-phosphate dehydrogenase. The deficiency of this enzymatic activity, either genetically transmitted or induced by dehydroepiandrosterone, is coupled with a reduced rate of benzo(a)pyrene conversion to water-soluble metabolites by human skin fibroblasts.

Inhibition of promotion and persistent nodule growth by S-adenosyl-L-methionine in rat liver carcinogenesis: role of remodeling and apoptosis.

The resistant hepatocyte model (initiation/selection) and the triphasic model (initiation/selection followed by phenobarbital, for a maximum of 16 weeks) were compared for their ability to generate enzyme-altered foci (EAF) and nodules in the liver of Wistar rats initiated by diethylnitrosamine. The effects of S-adenosyl-L-methionine (SAM) on the development of preneoplastic tissue was tested in these experimental models. In the absence of phenobarbital (PB), EAF and early nodules (EN) went through a phase of rapid growth, between 4 and 9 weeks after initiation, to a phase in which progressive decrease in number and size occurred. By the 26th week only a few remodeling EAF and nodules were found. In PB-treated rats a rapid increase in the percentage of liver occupied by EAF and EN, up to the 9th week after initiation, was followed by a period of slow growth (from the 9th to the 20th week) and then, after PB withdrawal (20th week), by a drop in the number and size of EAF and EN. However, at the 26th week actively growing nodules with a low tendency to spontaneous remodeling (persistent nodules) developed. EAF and EN showed a high DNA synthesis 5 weeks after initiation. Thereafter, progressive decline in DNA synthesis, coupled with remodeling and decrease in number of biochemical markers, was seen both in the absence and, even though to a lesser extent, in the presence of PB, indicating that preneoplastic lesions became increasingly insensitive to PB. Relatively few apoptotic bodies could be observed in EAF and EN during PB treatment. After PB withdrawal, decrease in growth potential was coupled with increase in apoptotic bodies. In contrast, in persistent nodules relatively high apoptosis occurred which partially counterbalanced high DNA synthesis. Administration of SAM for a maximum of 16 weeks, starting at the 4th week after initiation, caused a great decrease in number and size of EAF and EN, associated with inhibition of DNA synthesis, high cell death by apoptosis, high remodeling, and loss of biochemical markers, in preneoplastic lesions of both PB-treated and untreated rats. A 1-8-week SAM treatment, started after the development of persistent nodules, caused a great regression of nodular lesions, coupled with a sharp fall in DNA synthesis and increase in apoptosis. It is suggested that inhibition by SAM of the development of preneoplastic tissue is linked to a shift of the equilibrium between cell production and cell death in favor of cell death.(ABSTRACT TRUNCATED AT 400 WORDS)

Chemoprevention of rat liver carcinogenesis by S-adenosyl-L-methionine: a long-term study.

Previous work has shown a consistent fall in S-adenosyl-L-methionine (SAM) in the liver of diethylnitrosamine-initiated rats, during the development of preneoplastic lesions, in persistent nodules (PNs), and hepatocellular carcinomas. The injection of SAM into rats causes the reconstitution of the SAM pool, coupled with growth restraint, remodeling, and apoptosis of preneoplastic cells, and inhibits the development of PNs and hepatocellular carcinomas. To evaluate if SAM treatment causes a long-term prevention of preneoplastic and neoplastic liver lesions or merely causes a delay in their development, we evaluated the effect of a relatively short SAM treatment on the development of preneoplastic and neoplastic lesions in a long-term study. Male Wistar rats were subjected to initiation with diethylnitrosamine, followed by selection and then by the administration of phenobarbital for 16 weeks. After selection, the rats were given i.m. injections of a purified SAM preparation (384 mumol/kg/day) for 24 weeks. In SAM-treated rats, a decrease in the incidence of PNs was found 6, 14, and 24-28 months after initiation. At the end of SAM treatment the number of PNs per rat liver, nodule diameter, and labeling and mitotic indices of nodular cells decreased considerably in control rats. Nodule diameter started to increase rapidly again only 8 months after arresting SAM treatment, when complete recovery of DNA synthesis in nodular cells occurred. The majority of nodules present in the liver 6-28 months after initiation belonged to the clear and acidophilic cell types, with lower percentages of mixed cell and basophilic cell types. A decrease in basophilic nodules occurred in SAM-treated rats. Fourteen and 24-28 months after initiation hepatocellular carcinoma incidence was 11 of 12 and 10 of 10 in control rats, respectively, and only 1 of 12 and 3 of 11 in SAM-treated rats. At the 24th-28th month all control rats had tumors identified as 2 poorly differentiated carcinomas, 6 trabecular carcinomas, or 3 adenocarcinomas, while only 2 relatively small trabecular carcinomas and 1 small glandular tumor developed in SAM-treated rats. In 3 of 11 SAM-treated rats, but in none of the control rats, leukemic infiltration of liver occurred 24-28 months after initiation. Leukemic infiltration of the spleen occurred in 5 and 3 control and SAM-treated rats, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Reversal by 5-azacytidine of the S-adenosyl-L-methionine-induced inhibition of the development of putative preneoplastic foci in rat liver carcinogenesis.

The development of gamma-glutamyltranspeptidase (GGT)-positive foci, in Wistar rats, initiated with diethylnitrosamine and subjected to selection according to 'resistant hepatocyte' protocol, was coupled, 7 weeks after initiation, with liver DNA hypomethylation and with a fall in S-adenosylmethionine/S-adenosylhomocysteine (SAM/SAH) ratio, and in 5-methylthio-adenosine (MTA) content. A 15-day treatment with SAM, started 1 week after selection, caused a dose-dependent decrease in the development of GGT-positive foci, recovery of liver SAM/SAH ratio and MTA level, and liver DNA methylation. A 12-day treatment with 20 mumol/kg per day of 5-azacytidine (AzaC), starting 1 week after selection, enhanced growth of GGT-positive foci, caused strong DNA hypomethylation, and partially counteracted the inhibition of GGT-positive foci growth, without affecting recovery of SAM/SAH ratio and MTA level, induced by SAM. These results suggest a role of DNA methylation in the antipromoting effect of SAM.

Correlation between S-adenosyl-L-methionine content and production of c-myc, c-Ha-ras, and c-Ki-ras mRNA transcripts in the early stages of rat liver carcinogenesis.

gamma-Glutamyltranspeptidase (GGT)-positive and glutathione S-transferase (placental-GST-P) positive foci were induced in male Wistar rats by initiation with diethylnitrosamine (DENA), followed by selection and phenobarbital (PB). GGT- and GST-P-positive foci occupied 20-46% and 27-68% of liver parenchyma, respectively, 5-9 weeks after initiation. A high DNA synthesis was found in GGT-positive foci. Decrease in S-adenosyl-L-methionine (SAM) level and SAM/S-adenosylhomocysteine (SAH) ratio, and overall DNA hypomethylation occurred in the liver during the development of enzyme altered foci (EAF). These parameters underwent very small and transient changes in the liver of uninitiated rats at the 5th week, when EAF occupied 0.7-1.4% of the liver. At the 9th week, high RNA transcripts of c-myc, c-Ha-ras, and c-Ki-ras were found in the liver of initiated rats, but not in that of uninitiated rats. Immunohistochemical evaluation of c-myc gene product showed overexpression in GST-P-positive cells. SAM treatment of initiated rats caused inhibition of EAF growth, recovery of SAM/SAH ratio and DNA methylation, and decrease in protooncogene expression proportional to the dose and length of treatment. Liver SAM/SAH ratio was positively correlated with DNA methylation, and negatively correlated with transcript levels of the three protooncogenes. Thus, decrease in SAM/SAH ratio and DNA hypomethylation are early features of hepatocarcinogenesis promotion in rats fed a diet containing adequate lipotrope amounts, paralleled by overexpression of growth-related genes and rapid growth. Re-establishment of a physiologic SAM level makes it possible to inhibit protooncogene expression and EAF growth and to prevent late liver lesion development.

Comparative effects of L-methionine, S-adenosyl-L-methionine and 5'-methylthioadenosine on the growth of preneoplastic lesions and DNA methylation in rat liver during the early stages of hepatocarcinogenesis.

Male Wistar rats, initiated with diethylnitrosamine (DENA), were subjected to a selection treatment, according to the "resistant hepatocyte" model, followed or not followed by phenobarbital (PB). Rats received, for 3 weeks after selection, 4 i.m. doses (96 mmol/kg) of L-methionine, S-adenosyl-L-methionine (SAM), or 5'-methylthioadenosine (MTA), a SAM catabolite formed during polyamine synthesis or by spontaneous splitting of SAM at physiologic temperature and pH. They were then killed. In some rats, SAM and MTA treatments were started 20 weeks after initiation. The animals were killed 3 weeks later and persistent (neoplastic) nodules (PN) were collected. Some rat groups received 1/2 and 1/4 of the above SAM and MTA doses, or 1/8 of the above MTA dose. SAM and MTA, but not methionine, caused a dose-dependent decrease in number and surface area of gamma-glutamyltranspeptidase (GGT)-positive foci, and in labeling index (LI) of focal cells, coupled with remodeling. SAM and MTA liver contents, SAM/S-adenosylhomocysteine (SAH) ratio and overall methylation of liver DNA were low during the development of GGT-positive foci. SAM, but not methionine, caused a dose-dependent recovery of SAM content and DNA methylation, and a partial reconstitution of liver MTA pool. Exogenous MTA only induced the reconstitution of MTA pool, without affecting SAM level and DNA methylation. Recovery of SAM and MTA pool and DNA methylation was found in the rats subjected to SAM plus MTA, indicating the absence of inhibition of DNA methyltransferases in vivo by MTA. MTA also inhibited liver reparative growth in partially hepatectomized rats, without modifying SAM content and DNA methylation of regenerating liver (RL). A high activity of ornithine decarboxylase (ODC) was found in the liver, during the development of preneoplastic foci, and in PN. This activity was inhibited by SAM and MTA treatments. Although MTA was more effective than SAM, the decrease in ODC activity was coupled with a larger fall in DNA synthesis in SAM-treated than in MTA-treated rats. Thus the antipromotion effect of SAM could not merely depend on its (spontaneous) transformation into MTA. Although MTA production may play a role in the SAM antipromotion effect, other mechanisms could be involved. A role of DNA methylation in the inhibition of growth by SAM is suggested. MTA is a potential chemopreventive agent for liver carcinogenesis.

Effect of S-adenosyl-L-methionine on the development of preneoplastic foci and the activity of some carbohydrate metabolizing enzymes in the liver, during experimental hepatocarcinogenesis.

gamma-Glutamyltranspeptidase (GGT)-positive foci and glutathione-S-transferase, placental (GST-P)-positive lesions occupied 36% and 54% of liver parenchyma, respectively, in Wistar rats 8 weeks after initiation with diethylnitrosamine, followed by selection. The administration of S-adenosyl-L-methionine (SAM, 384 mumol/kg/day) caused 77% and 42% falls in the percentage of GGT-positive and GST-P-positive lesions, respectively. There also occurred a 46% decrease in labeling index of GGT-positive foci, in SAM-treated rats. These changes were associated with decrease in liver pyruvate kinase (PK), lactate dehydrogenase and glycerol-3-phosphate dehydrogenase. SAM did not affect these enzymatic activities in normal and uninitiated controls, but it caused a consistent increase in initiated rats. Enolase, fructose-biphosphatase and malic enzyme (ME) activities increased in the liver of initiated rats. SAM did not modify significantly these enzymatic activities, either in control or in initiated rats. Glucose-6-phosphate dehydrogenase (G6PDH) was 113% higher in the liver of initiated rats than in uninitiated controls. SAM treatment did not significantly affect this enzymatic activity in uninitiated rats, but caused a great decrease in initiated ones. As expected, there occurred a marked rise in GGT activity in the liver of initiated rats, with respect to controls. SAM caused an increase in GGT activity in normal and uninitiated controls, but it caused a 77% fall in GGT activity in initiated rats, coupled with a 380% rise in remodeling of GGT-positive lesions. Histochemical determination of G6PDH and ME activities showed that in the absence of SAM many preneoplastic lesions expressed higher G6PDH and ME activities than surrounding liver. SAM did not affect ME-positive lesions, while it caused a decrease in the number of G6PDH-positive lesions. Immunohistochemical determination of PK activity, isoenzyme L, showed a decrease in GST-P-positive lesions. Many of these lesions were no longer recognizable as lesions expressing a low PK activity, in SAM-treated rats. However, a relatively small number of GST-P-positive lesions expressing a low PK activity were still present in these rats. These data suggest that glucose channelled into triacylglycerol and pyruvate synthesis decreases in rat liver, during the development of preneoplastic foci, while the production of reducing equivalents and pentose phosphates increases, thus favoring DNA synthesis and detoxification reactions. Decrease in DNA synthesis, in SAM-treated rats, is paralleled by a partial reversion of carbohydrate metabolic features to those present in normal liver.

Modulatory mechanisms of chemical carcinogenesis: the role of the NADPH pool in the benzo(a)pyrene activation.

Human lymphocytes and human skin fibroblasts isolated in vitro from subjects carrying the Mediterranean variant of glucose-6-phosphate dehydrogenase (G6PD) exhibit an 86-87% decrease of this enzymatic activity. This is coupled with 51% and 61% decreases of the NADPH/NADP+ ratio in the G6PD-deficient human lymphocytes (HL) and human skin fibroblasts (HSF), respectively. There also occurs a 63-67% decrease of the hexose monophosphate shunt (HMS) in the deficient cells. Incubation with 0.1 mM methylene blue stimulates the HMS of normal HL 15-fold and that of deficient lymphocytes only 2.4-fold. These figures are, respectively, 7 and 2.2 in the case of HSF. This behavior of G6PD-deficient HL and HSF is coupled with an increase of the resistance to the cell death induced by benzo(a)pyrene (BP). This effect is mimicked by the incubation of normal HSF with dehydroepiandrosterone (DEA) which strongly inhibits G6PD. In contrast, no differences between normal and deficient HSF occur as a result of the effect of methylnitrosourea (MNU), a carcinogen that does not need metabolic activation. The NADPH-cytochrome c (P450) reductase of G6PD-deficient HL and HSF homogenates becomes lower than that of controls when endogenous G6PD and exogenous glucose 6-phosphate (G6P) and NADP+ are used as a hydrogen donor system in place of NADPH. Normal and G6PD-deficient HL, having comparable BP-hydroxylating activities, in the presence of exogenous G6P, NADP+, and G6PD, were studied to determine the effect of the absence of exogenous G6PD in the reaction system.(ABSTRACT TRUNCATED AT 250 WORDS)

The variations of S-adenosyl-L-methionine content modulate hepatocyte growth during phenobarbital promotion of diethylnitrosamine-induced rat liver carcinogenesis.

A decrease in liver S-adenosyl-L-methionine (SAM) content and an increase in ornithine decarboxylase (ODC) activity occurred between the 2nd and the 5th week after starting 2-acetylaminofluorene (AAF) feeding in diethylnitrosamine (DENA)-initiated rats. These rats then received a 0.05% phenobarbital (PB)-containing diet for 18 weeks after the end of AAF feeding. Two weeks after starting AAF, an increase in the hepatocyte labeling index (LI) also occurred in gamma-glutamyl-transpeptidase (GGT)-positive foci and surrounding tissue. LI returned to control values in a few days in surrounding tissue, while it remained high for at least 4 weeks in the foci. Analogous changes were observed, but for a shorter period of time, in the rats subjected to partial hepatectomy (PH) plus AAF, in which no GGT-positive foci developed. Twenty-four weeks after starting AAF, 30% of the liver was occupied by visible nodules. ODC activity and LI were high and SAM was low in nodules, but they were near to control values in surrounding liver. SAM administration reconstituted the liver SAM pool, inhibited ODC activity, and prevented visible nodule development. SAM inhibition of ODC activity occurred in vitro only after preincubation with liver homogenate and was enhanced by adenine, an inhibitor of methylthioadenosine (MTA) phosphorylase. MTA addition to the reaction of mixture for ODC determination was inhibitory. The SAM decrease in both liver and nodules was coupled with a decrease of MTA content. SAM administration caused MTA accumulation in the liver. It is suggested that liver SAM content by influencing MTA level, could be a rate-limiting factor for growth and promotion, through a modulation of polyamine synthesis.

Inhibition by ethanol of rat liver plasma membrane (Na+,K+)ATPase: protective effect of S-adenosyl-L-methionine, L-methionine, and N-acetylcysteine.

(Na+,K+)ATPase activity of rat liver plasma membranes was evaluated in female rats feeding an ethanol containing diet for 46 days (total ethanol ingested, 59.7 g/100 g body wt). Determinations were performed at the end of ethanol treatment or at various times after stopping treatment. (Na+,K+)ATPase and 5'-nucleotidase activities exhibited a 8- and 1.4-fold decrease, respectively, at the end of ethanol ingestion. In contrast no modifications of Mg2+-ATPase activity were observed. There also occurred, in ethanol-treated rats, release of sorbitol dehydrogenase into the blood, fat accumulation in liver cells, and decrease in reduced glutathione (GSH) liver content. A decrease in (Na+,K+)ATPase activity was also found in plasma membranes isolated from hepatocyte suspensions after a 2-hr incubation with 50 mM ethanol or 1 mM acetaldehyde (ACA), in conditions that caused a great fall in hepatocyte GSH content but did not cause cell death. After the cessation of ethanol administration, there occurred a progressive recovery of (Na+,K+)ATPase activity, GSH and triacylglycerol content, and release of sorbitol dehydrogenase. These parameters reached control values 12 hr after ethanol withdrawal. S-Adenosyl-L-methionine (SAM), L-methionine, and N-acetylcysteine (NAC), given to rats during ethanol treatment, prevented the decrease in (Na+,K+)ATPase activity and GSH content. They also reduced steatosis and liver necrosis. The efficiency of these compounds decreased in this order: SAM, methionine, NAC. SAM accelerated the recovery of all parameters studied after ethanol withdrawal, and also protected (Na+,K+)ATPase activity and GSH content of isolated hepatocytes from the deleterious effect of ethanol. These SAM effects were prevented by 1-chloro-2,4-dinitro-benzene, a compound which depletes cell GSH. Treatment of isolated hepatocytes with [35S]SAM led to the synthesis of labeled GSH. The total amount and specific activity of labeled GSH underwent a significant increase, in the presence of 2 mM ethanol or 0.5 mM ACA, which indicates a marked stimulation of GSH synthesis by ethanol and ACA. These data indicate that ethanol intoxication may inhibit (Na+,K+)ATPase activity; an effect that does not seem to depend on cell necrosis. SAM, methionine, and NAC exert various degrees of protection toward ethanol-induced cell injury, which are related to the efficiency of these compounds in maintaining a high GSH pool.

Early stimulation of polyamine biosynthesis during promotion by phenobarbital of diethylnitrosamine-induced rat liver carcinogenesis. The effects of variations of the S-adenosyl-L-methionine cellular pool.

A decrease of S-adenosyl-L-methionine liver content was observed between the 14th and the 35th day after the start of 2-acetylaminofluorene feeding in diethylnitrosamine-initiated rats according to the 'resistant-hepatocyte' model of hepatocarcinogenesis. The decrease was enhanced by phenobarbital given to the animals after the end of 2-acetylaminofluorene feeding. These changes were associated with an increase in ornithine decarboxylase activity and the spermidine:spermine ratio. S-adenosyl-L-methionine administration to rats caused a great fall in the percentage of gamma-glutamyltranspeptidase-positive liver as well as in polyamine synthesis. An increase in ornithine decarboxylase activity, associated with a decrease in the liver S-adenosyl-L-methionine pool, also occurred in normal animals on the first day following a partial hepatectomy and was enhanced by phenobarbital. The association of 2-acetylaminofluorene feeding with partial hepatectomy resulted in a slower liver regeneration, while the decrease in S-adenosyl-L-methionine level and the increase in polyamine synthesis were observed over a longer period of time after partial hepatectomy. These changes were further prolonged in diethylnitrosamine-initiated rats in which gamma-glutamyltranspeptidase-positive foci developed. In these animals a high level of polyamine synthesis was still present when liver regeneration was complete. At this stage of the observation period the labeling index was very low in surrounding liver, but still high in the gamma-glutamyltranspeptidase-positive areas. Phenobarbital stimulated polyamine synthesis and cell growth and further prolonged the period of time during which a high ornithine decarboxylase activity and labeling index were present. These results indicate that the liver lipotrope content could be a rate-limiting factor for cell growth and liver neoplasia promotion and this could depend on the modulation of polyamine biosynthesis.

Inhibition by dehydroepiandrosterone of liver preneoplastic foci formation in rats after initiation-selection in experimental carcinogenesis.

The effect of dehydroepiandrosterone (DHEA) on the development of liver preneoplastic foci was evaluated. The experimental protocol used initiation by diethylnitrosamine (DENA), followed by selective growth induced by partial hepatectomy (PH), in rats fed an N-acetylamino-fluorene (AAF)-containing diet, and followed by a standard diet with or without 0.05% phenobarbital (PB). DHEA (0.6%) was administered in the diet for 4 or 7 weeks after DENA injection (treatments A and B) or for 3 weeks after the end of AAF feeding (treatment C). On the 7th week after DENA injection, DHEA treatments A and C caused slight decrease of body weight and 40-60% increase in liver weight and cell size; number of nuclei per g of liver was not changed. The dietary treatment also caused a marked decrease of liver glucose-6-phosphate dehydrogenase (G6PD) activity and of the percentage of the gamma-glutamyltranspeptidase (GGT)-positive liver. PB increased G6PD activity and GGT-positive liver. This effect was oblated by DHEA treatments A and C. On the 7th week, the labeling index (LI) was low in surrounding liver, and high in GGT-positive foci. PB had an enhancing effect, while DHEA treatments A and C were inhibitory. G6PD was low at the end of DHEA treatment B, but it returned to normal values 4 weeks after the end of this treatment. At this time no effect of DHEA treatment B was observed on the extent of GGT-positive liver and LI.(ABSTRACT TRUNCATED AT 250 WORDS)

Benzo(a)pyrene metabolism by lymphocytes from normal individuals and individuals carrying the Mediterranean variant of glucose-6-phosphate dehydrogenase.

In vitro growing human lymphocytes (HL) and fibroblasts, isolated from glucose-6-phosphate dehydrogenase (G6PD)-deficient subjects (Mediterranean variant), show a sharp decrease in this enzymatic activity and in NADPH:NADP+ ratio. These cells are less able than controls to hydroxylate benzo(a)pyrene (BaP) when tested in the absence of an exogenous NADPH-generating system. They exhibit great resistance to the toxic effect of BaP. G6PD-deficient fibroblasts are less prone than controls to in vitro transformation by BaP. To investigate whether this depends on a decreased production of active BaP metabolites and BaP:DNA adducts by G6PD-deficient cells, BaP metabolism was studied in G6PD-deficient HL cultured in vitro in the presence of mitogens and treated with BaP for 24 hr. HPLC profiles of organo- and water-soluble metabolites revealed that both types of benzo(a)anthracene (BaA)-induced HL produced: 4,5-, 7,8-, 9,10-diols, 1,3-, 3,6-quinones, 3-, 9-hydroxy and 2 peaks of more polar metabolites. There was a 25-76% decrease of organo- and water-soluble metabolites in the G6PD-deficient cells. When HL were incubated with 7,8-diol, the formation of metabolites mutagenic for Salmonella typhimurium (His-) was very low in G6PD-deficient cells. BaP:deoxyadenosine (dAde) and BaP:deoxyguanosine (dGua) adducts were identified after incubation of both types of HL with BaP. There was a 31-79% fall in adduct formation by G6PD-deficient cells. Our results indicate that G6PD-deficient human lymphocytes are less able to metabolize BaP than normal lymphocytes. We suggest that the NADPH pool is inadequate, in deficient cells, for active BaP metabolism.

Protooncogene methylation and expression in regenerating liver and preneoplastic liver nodules induced in the rat by diethylnitrosamine: effect of variations of S-adenosylmethionine:S-adenosylhomocysteine ratio.

S-adenosylmethionine:S-adenosylhomocysteine (SAM/SAH) ratio, 5-methylcytosine (5mC) DNA content, and methylation and expression of c-myc, c-Ha-ras and c-Ki-ras have been studied in liver nodules, induced by diethylnitrosamine according to the 'resistant hepatocyte' model, and in regenerating liver (RL) between 0.5 and 72 h after partial hepatectomy (PH). Nodules, 11, 13 and 21 weeks after initiation, grew actively, showed a low tendency to remodel (persistent nodules), and did not exhibit carcinomatous changes. They underwent extensive remodeling after a 1-week SAM treatment (64 mumol/kg/day), and decreased in size and number after a 3-11-week treatment. A low SAM/SAH ratio was coupled, in nodules, with a high labeling index (LI), 2-fold fall in 5mC DNA content, increase in c-myc, c-Ha-ras and c-Ki-ras expression and hypomethylation of CCGG sequences in the DNA hybridizing with the three protooncogenes. In RL a low SAM/SAH ratio, overall DNA hypomethylation and enhanced c-myc expression were first observed 0.5 h after PH, reached a peak at 5 h and progressively returned to pre-PH levels later on. Maximum expression of c-Ha-ras and c-Ki-ras occurred 24-30 h after PH, roughly coincident with the LI peak. However, no great modifications of the methylation pattern of protooncogene CCGG sequence occurred at any time after PH, indicating the presence of hypomethylated genes and/or DNA sequences different from those investigated in this paper. SAM injection to nodule-bearing rats, for 1-11 weeks before killing, and to hepatectomized rats, 2 days before PH and then up to killing, largely prevented decrease in the SAM/SAH ratio and overall DNA methylation and inhibited LI and protooncogene expression. In nodules these effects were proportional to the treatment length and coupled with methylation of CpG residues in the CCGG sequence of the three protooncogenes studied. SAM treatment left the methylation pattern of these genes unchanged in RL. Kinetics of increase in protooncogene expression suggest a role in the regulation of cell cycle in RL. However, decrease in the SAM/SAH ratio, protooncogene hypomethylation and enhanced expression are apparently stable in nodules 11-21 weeks after initiation and could be implicated in continuous nodule growth and progression. Control of DNA methylation and gene expression by exogenous SAM could be a mechanism of the SAM anti-progression effect.

Dependence of benzo(a)pyrene metabolism on NADPH pool in normal and glucose-6-phosphate dehydrogenase deficient human fibroblasts.

Human skin fibroblasts (HSF), from normal donors and donors carrying the Mediterranean variant of glucose-6-phosphate dehydrogenase, grown in vitro in the presence of 0.25-5 microM benzo(a)pyrene (BaP), produced the following organic-soluble metabolites: 9,10-diol, 7,8-diol, quinones, 3- and 9-hydroxide and a more polar fraction, and the following water-soluble metabolites: more polar, 3- and 9-hydroxide and 9,10-diol. Single organic- and water-soluble metabolites increased with BaP concentration in both types of HSF, but the ratio normal/variant increased with BaP concentration. NADPH level and NADPH/NADP+ ratio underwent a slight decrease in normal HSF incubated with 2.5 microM BaP, while a greater fall occurred in the deficient HSF at 0.25 and 2.5 microM BaP. NADPH content seems to be rate-limiting for BaP metabolism in the deficient cells.

Alterations of ornithine decarboxylase gene during the progression of rat liver carcinogenesis.

Liver nodules and carcinomas, developing in F344 rats initiated with diethylnitrosamine, exhibit high ornithine decarboxylase (ODC) activity and DNA synthesis. ODC-related RNAs of 1.8, 2.1 and 2.6 kb are produced by normal rat liver. Early preneoplastic nodules, developing 10 weeks after initiation, showed overproduction of 1.8 and 2.1 kb RNAs, while the 2.6 kb RNA was barely detectable. Rises in the 1.8, 2.1 and 2.6 kb RNAs occur in late nodules (30 weeks after initiation) and in carcinomas. The comparison of different tissues for relative increase in ODC activity, RNA levels and DNA synthesis showed that these parameters behaved in the same way: highest increases occurred in early nodules and carcinomas. These observations suggest that overexpression of ODC gene and alterations in regulatory mechanisms of ODC gene expression may be implicated in the progression of preneoplastic lesions to malignancy. Southern blot analysis of PstI DNA digests revealed the presence of ODC gene rearrangement in two carcinomas and in one late nodule. However, the role of this phenomenon in the progression of preneoplastic lesions is unclear, due to the possibility that ODC pseudogenes are involved instead of or in addition to ODC gene.

Reversal by ribo- and deoxyribonucleosides of dehydroepiandrosterone-induced inhibition of enzyme altered foci in the liver of rats subjected to the initiation--selection process of experimental carcinogenesis.

The effect of dehydroepiandrosterone (DHEA) on the activity of NADPH-producing enzymes and the development of enzyme-altered foci has been investigated in the liver of female Wistar rats subjected to an initiating treatment (a necrogenic dose of diethylnitrosamine) followed, 15 days later, by a selection treatment [a 15-day feeding of a diet containing 0.03% 2-acetylaminofluorene (2-AAF), with a partial hepatectomy at the midpoint of this feeding]. At the end of the selection treatment all rat groups received, for 15 days, a basal diet containing, when indicated, 0.05% phenobarbital (PB) and/or 0.6% DHEA. The effect of DHEA on the activity of NADPH-producing enzymes was also studied in normal rats fed, for 15 days, a diet containing 0.6% DHEA and in their pair-fed controls. DHEA caused a 43-58% inhibition of glucose-6-phosphate dehydrogenase (G6PD) and, respectively, 338-420% and 21-24% increases in malic enzyme (ME) and isocitric dehydrogenase activities in all rat groups. This was coupled with a great fall in the production of ribulose-5-phosphate, while no change in NADP+/NADPH ratio occurred. Hepatocytes, isolated from DHEA-treated rats, exhibited a very low activity of hexose monophosphate shunt (HMS), which was not stimulated by methylene blue, an exogenous oxidizing agent that markedly stimulated HMS activity in control hepatocytes. DHEA caused a great fall in the percentage of liver occupied by gamma-glutamyltranspeptidase (GGT)-positive foci, in the rats subjected to the initiation-selection treatments. PB enhanced the development of these foci, an effect which was completely overcome by DHEA. In addition, focal cells no longer expressed a G6PD activity higher than that of surrounding liver in DHEA-treated rats, but exhibited a high histochemical reaction for ME. DHEA also caused a great fall in labelling index of GGT-positive foci. Starting at the end of 2-AAF feeding, a mixture of ribonucleosides (RNs) of adenine, cytosine, guanine and uracil and of deoxyribonucleosides (DRNs) of adenine, cytosine, guanine and thymine were injected i.p. every 8 h for 12 days to the rats subjected to the initiation-selection treatments plus PB. Rats were killed 3 days after the end of RN and DRN treatments. These treatments completely overcome the DHEA effect on the development of GGT-positive foci and DNA synthesis by the focal cells, without affecting G6PD activity of both whole liver and putative preneoplastic foci. Experiments with labeled nucleosides revealed that RNs and DRNs produced derivatives that were incorporated into liver DNA.(ABSTRACT TRUNCATED AT 400 WORDS)

Decreased stimulation by 12-O-tetradecanoylphorbol-13-acetate of superoxide radical production by polymorphonuclear leukocytes carrying the Mediterranean variant of glucose-6-phosphate dehydrogenase.

Polymorphonuclear leukocytes (PMNs) from individuals carrying the Mediterranean variant of glucose-6-phosphate dehydrogenase (G6PD) exhibit a great decrease in this enzymatic activity and in hexose monophosphate shunt (HMS). 12-O-tetradecanoylphorbol-13-acetate (TPA) greatly stimulates HMS of normal PMNs, while it does not affect that of the deficient PMNs. Similarly, the stimulation of HMS by methylene blue is largely reduced in G6PD-deficient PMNs. These changes are paralleled by a 58% decrease in TPA-stimulated superoxide radical (O2-) formation by the deficient PMNs. G6PD activity is not detectable in the deficient PMNs incubated with dehydroepiandrosterone, and these cells show a near complete inhibition of O2- production. It thus seems that the low ability of G6PD-deficient PMNs in the production of O2- depends on the low NADPH generation by HMS in these cells. The decrease in TPA-stimulated O2- production suggests a reduced response of G6PD-deficient cells to promoting agents.

Effect of glucose-6-phosphate dehydrogenase deficiency on the benz(a)pyrene toxicity for in vitro cultured human skin fibroblasts.

Human skin fibroblasts, isolated in vitro, from donors carrying the Mediterranean variant of glucose-6-phosphate dehydrogenase, exhibit a sharp decrease of hexose monophosphate shunt and NADPH/NADP+ ratio when compared to the fibroblasts from normal donors. This behavior is coupled to an increase of the resistance to cell death and growth inhibition induced by benz(a)pyrene, whose activation proceeds through the NADPH-dependent arene oxide formation. No differences were observed in the toxic effects of methylnitrosourea, a carcinogen that does not need metabolic activation, on normal and variant fibroblasts.