Maria E. Ruggiu

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 γ-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. The observation that an inhibition of GGT-positive liver formation occurs when DENA is given at the end of the AAF/PH treatment in DENA initiated rats could indicate that the DHEA antipromoting effect depends more on growth inhibition than on interference with carcinogen metabolism.

Control of Glucose-6-Phosphate Dehydrogenase Deficiency on the Formation of Mutagenic and Carcinogenic Metabolites Derived from Benzo(a)pyrene

It has been observed that human lymphocytes (HL) and fibroblasts, isolated in vitro from donors carrying the Mediterranean variant of glucose-6-phosphate dehydrogenase (G6PD), show a great decrease in this enzymatic activity, the hexose monophosphate shunt, and the NADPH/NADP+ ratio. This effect is associated with a decreased sensitivity of G6PD-deficient cells to the benzo(a)pyrene (BaP) cytotoxic effect and to a decreased in vitro transformation of BaP-treated fibroblasts. Further, benzo(a)anthracene (BaA)-induced BaP hydroxylase activity is lower in G6PD-deficient cells, when measured in the presence of endogenous NADPH. It has been hypothesized that the NADPH level could be rate-limiting for the NADPH-dependent steps of BaP metabolic activation. To test this hypothesis, the formation of BaP metabolites was studied in normal and G6PD-deficient HL incubated with the carcinogen. HPLC profiles of organic-soluble metabolites revealed that both types of HL produced all the following known BaP metabolites: 9,10-, 4,5- and 7,8-dihydrodiols, quinones, 9- and 3-hydroxy and two peaks of more polar metabolites. There was a great decrease of the various metabolites in the deficient HL. A decrease of total water-soluble BaP metabolites also occurred. HL formed mutagenic metabolites for S. typhimurium TA100 (his-) when incubated with the rat liver S9 fraction. When intact HL substituted S9 fraction, a significant reversed mutation occurred only with normal HL. This could indicate that the NADPH pool is inadequate in G6PD-deficient HL for active BaP metabolism. Accordingly, deficient HL formed lower amounts of BaP:DNA adducts than control during incubation with BaP.

S-Adenosylmethionine Antipromotion and Antiprogression Effect in Hepatocarcinogenesis. Its Association with Inhibition of DNA Methylation and Gene Expression

The identification of several steps in the carcinogenic process, has permitted a new approach in the prevention of cancer development. It is conceivable that interference with one or more of the recognizable steps leads to the breakdown of the carcinogenic process. In recent years some attempts to interfere with carcinogenic promotion, at different levels, have been made using retinoids1,2, 1α, 25-dihydroxyvitamin D3, a hormonally active metabolite of vitamin D3, antioxidants4,5, α-difluoromethylornithine6,7, putrescine8, indomethacin9, protease inhibitors10, inhibitors of arachidonic acid metabolism11, protein kinase C inhibitors12, dehydroepiandrosterone and some related hormones13–16, and S-adenosylmethionine17–21 (SAM).

Inhibition of Initiation and Promotion Steps of Carcinogenesis by Glucose-6-Phosphate Dehydrogenase Deficiency

A number of observations indicate that deficiency of glucose-6-phosphate dehydrogenase (G6PD), either genetically transmitted or caused by dehydroepiandrosterone (DHEA)1, or some related steroids, is associated with an antitumor effect. Some epidemiologial evidence of a decreased tumor incidence in G6PD-deficient subjects has been reported2–5. Although these observations, do not definitively prove the existence of clear relationships between G6PD-deficiency and tumor incidence, they suggest a negative correlation. In accordance with this hypothesis retrospective studies have shown subnormal plasma levels of DHEA or DHEA-sulfate in women with breast cancer6,7. Moreover, in a prospective study a subnormal urinary excretion of the DHEA metabolites androsterone and etiocholanolone has been found to be associated with enhanced breast cancer risk8. DHEA mimics many of the effects of the genetically transmitted G6PD deficiency as concerns the resistance of in vitro cultured cells to the toxic and transforming effects of carcinogens. Following the first observation by Schwartz9 that long-term per os treatment with DHEA inhibits the formation of spontaneous tumors in mice, different other studies have shown that this treatment also prevents the development of chemically induced tumors in the same animal10–13, as well as in the rat14. DHEA is known to exhibit an anti-obesity effect (cf. ref. 15 for review). Even if in some rat strains DHEA causes a decrease in food intake, it has been suggested that the anti-obesity effect is linked to a decreased food utilization more than to a reduced intake16. Alternatively, an increased s-oxidation of fatty acids in peroxisomes, could represent an “energy wasting” pathway in DHEA-treated animals17. Reduction in food intake is known to decrease the susceptibility of different tissues to cancer18. However, the possibility that the DHEA anti-tumor action is linked to food restriction, is ruled out by the observation that topic application of DHEA, while not reducing body weight, greatly inhibits development of skin tumor in mice12.

Chemoprevention of Rat Liver Carcinogenesis by S-Adenosylmethionine: Role of Remodeling and Apoptosis

A great deal of evidence indicates that modification of methyl donor liver content and methylation reactions play a role in hepatocarcinogenesis. The original Copeland and Salmon’s experiments1 showing the development of hepatocellular carcinomas in rats chronically fed a choline-deficient diet, even if difficult to interpret, because of the possible presence of aflatoxin B1 and other carcinogenic contaminants in the diet, focused the interest on the role of labile methyl group deficiency in carcinogenesis. This role has been confirmed by the observation that ethionine an antagonist of the methyl group donor methionine, causes cancer2. In addition, methyl-deficient diet was found to enhance several biochemical effects as well as transforming activity of various carcinogens3. Recent experiments from three different laboratories in which methyl-deficient diets and rat’s environment have been accurately checked for relevant contamination with chemical carcinogens, have definitely proved that chronic feeding of these diets, causes the development of rat liver cancer4–6. However, even if the carcinogenicity of methyl-deficient diet has been well proved, it has not been definitely established if methyl deficiency may be considered a complete carcinogen7 or a promoter8. Further details on these and other aspects of tumor induction by methyl deficiency may be found in various comprehensive reviews published in the recent years8–10.

Relationships between NADPH Content and Benzo(a)pyrene Metabolism in Normal and Glucose-6-Phosphate Dehydrogenase-Deficient Human Fibroblasts

Previous work in our laboratory has shown that human skin fibroblasts (HSF) and human lymphocytes carrying the Mediterranean variant of glucose-6-phosphate dehydrogenase (G6PD) exhibit a great decrease in hexose monophosphate shunt and in NADP /NADPH ratio1,2. G6PD deficiency protects in vitro growing HSF and lymphocytes from benzo(a)pyrene (BaP) toxicity. G6PD-deficient HSF give rise in soft agar, after incubation with BaP, to a lower number of colonies than normal HSF1. Aryl hydrocarbon hydroxylase (AHH) activities are lower in G6PD-deficient cells, when tested in the absence of exogenous NADPH2. G6PD-deficient cells, incubated in vitro with BaP, produce low amounts of organic- and water-soluble BaP metabolites and show a decreased ability to form BaP-7,8-diol-9,10-epoxide and BaP-DNA adducts3,4.

S-Adenosylmethionine Content, DNA Methylation and Gene Expression in Regenerating Liver

Cell growth is a fundamental feature of various stages of experimental carcinogenesis. It is a prerequisite for initiation1, and one of the major events of the promotion and progression processes2.

Biochemical and Molecular Perturbations Induced in Preneoplastic Tissue by a S-Adenosyl-L-Methionine Load

A growing evidence indicates that labile methyl groups play an essential role in liver carcinogenesis. A depletion of these groups, in rats fed a lipotrope-deficient diet causes cancer or enhances liver carcinogenesis induced by various carcinogens1–3. Liver S-adenosyl-L-methionine (SAM) deficiency has also been demonstrated in rats fed a diet containing adequate amounts of lipotropes and subjected to a promoting treatment with phenobarbital (PB)4. Furthermore, a consistent fall in SAM liver content has been demonstrated in the liver, during the development of microscopic preneoplastic lesions, as well as in isolated preneoplastic nodules and carcinomas, developing in rats subjected to the initiation/promotion treatments of experimental hepatocarcinogenesis, and fed a diet supplemented with adequate amounts of lipotropic compounds5–9.