Serenella Frassetto

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.

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. When exogenous G6PD is omitted, the deficient HL exhibit a BP hydroxylase activity sharply lower than controls. G6PD-deficient HL and HSF are less prone than controls to produce BP water-soluble metabolites. A decrease in the synthesis of BP metabolites, mutagenic for his–Salmonella typhimurium in G6PD-deficient HL, has also been observed. These results suggest that the decreased susceptibility of G6PD-deficient cells and HSF to the toxic effect of BP and that of deficient HSF to its previously observed transforming effect, could depend on a reduced BP metabolism in the deficient cells.

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.

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.