Modulatory Mechanisms of Chemical Carcinogenesis: The Role of the NADPH Pool in the Benzo(a)pyrene Activation

Abstract

A EST RA CT Human lymphocytes and human skin fibroblasts isolated in vilro from subjects carrying the Mediterranean variant of glucose-6-phosphate dehydrogenase (CCPD) exhibit an 86-87% decrease of this enzymatic activity. This is coupled with 51% and 61% decreases of the NADPH/NADP+ ratio in the GCPD-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 CCPDdeficient 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 GCPD. 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 CCPD-deficient HL and HSF homogenates becomes lower than that of controls when endogenous GCPD and exogenous glucose 6-phosphate (CCP) and NADP' are used as a hydrogen donor system in place of NADPH. Normal and CCPD-deficient HL, having comparable BP-hydroxylating activities, in the presence of exogenous GCP, NADP+, and GCPD, were studied to determine the effect of the absence of exogenous GCPD in the reaction system. When exogenous CCPD is omitted, the deficient HL exhibit a BP hydroxylase activity sharply lower than controls. GCPD-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 CCPD-deficient HL, has also been observed.These results suggest that the decreased susceptibility of GCPD-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. Session I: 'Genetic Expression and Cell Transformation." October 12-15, Alghero. Italy. This symposium section will be continued in

Mechanism of inhibition of growth of 3T3-L1 fibroblasts and their differentiation to adipocytes by dehydroepiandrosterone and related steroids: role of glucose-6-phosphate dehydrogenase.

“…All of these effects may result from a limitation of intracellular NADPH supply stemming from a reduction in G6PD activity. Moreover, resistance to benzo[a]pyrene cytotoxicity can be mimicked in normal fibroblasts by treatment with DHEA (31)(32)(33)(34). Epidemiologic evidence also supports this hypothesis since individuals with G6PD deficiency may be more resistant to developing cancer than normal individuals from the same geographical area (for reviews, see refs.…”

“…Cells from individuals with the severe Mediterranean variant of G6PD deficiency show depression of metabolism, DNA-binding and cytotoxicity of benzo[a]pyrene. Furthermore, G6PD-deficient granulocytes generated lower amounts of superoxide in response to phorbol esters (31,32). All of these effects may result from a limitation of intracellular NADPH supply stemming from a reduction in G6PD activity.…”

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

“…Previous work in our laboratory has demonstrated that cultures of human skin fibroblasts (HSF) and human lymphocytes (HL) carrying the Mediterranean variant ofG6PD are more resistant than norma1 HSF and HL to the toxic effects of BaP (8)(9)(10)23). G6PD-deficient HSF appear to be less prone than normal cells to in vitro transformation by BaP, as assessed by the loss of anchorage-dependence (8).…”

“…G6PD-deficient HSF appear to be less prone than normal cells to in vitro transformation by BaP, as assessed by the loss of anchorage-dependence (8). It has been observed that G6PD-deficient HSF and HL exhibit a great decrease of the hexose monophosphate shunt, either stimulated or not by methylene blue, as well as of the NADPH/NADP+ ratio (8)(9)(10)23). Moreover, NADPH-cytochrome reductase and BaP hydroxylase activities were found to be lower in homogenates of G6PD-deficient HL, * Presented at the Third Sardinian International Symposium, when tested in a reaction system containing exogenow G6P and NADPf and endogenous G6PD.…”

“…G6PD-deficient Cells also produced lower amounts Of water-soluble and mutagenic metabolites after i~cubation with BaP (8,9)To further investigate BaP metabolism in G6PD-deficient HLY determined the Production of BaP metabo1ites and BaP:DNA adducts during the incubation of these cells with BaP.…”