Induction of oxidative DNA damage and early lesions in rat gastro-intestinal epithelium in relation to prostaglandin H synthase-mediated metabolism of butylated hydroxyanisole.

The effect of metabolic activation of the food additive 3-tert-butyl-4-hydroxyanisole (BHA) by prostaglandin H synthase on the gastro-intestinal cell proliferation was determined by studies of the nature and the time dependency of early lesions in the forestomach, glandular stomach and colon/rectum of rats given BHA with and without co-administration of acetylsalicyclic acid (ASA: an inhibitor of prostaglandin H synthase), in combination with the formation of oxidative DNA damage in the epithelial cells of glandular stomach and colon/rectum as well as in the liver. BHA appeared to be a strong inducer of oxidative DNA damage in the epithelial cells of the glandular stomach, increasing the level of 7-hydro-8-oxo-2'deoxyguanosine (8-oxodG) with increasing duration of BHA administration. Similar observations were made in colorectal DNA although levels of oxidative DNA damage tend to be smaller. In liver DNA, BHA appeared to be capable of increasing background 8-oxodG levels only after 14 days of treatment. This relatively slow response may be related to very low prostaglandin H synthase activity of liver cells. The severity of hyperplasia and inflammation in both forestomach and glandular stomach appeared to increase gradually with continued BHA administration. The hyperplasia induced by BHA was paralleled by inflammatory changes. In colorectal tissue, however, no tissue abnormalities were observed. This indicates that oxidative DNA damage induced by BHA is not a consequence of early lesions in gastro-intestinal epithelium, but might be the initial step in the stimulation of gastro-intestinal cell proliferation which, as shown previously, also occurs in colon epithelium. Co-administration of the prostaglandin H synthase inhibitor ASA resulted in a significant decrease of both epithelial oxidative DNA damage and the incidence of lesions, which indicates that this enzyme system is involved in the enhancement of cellular proliferation induced by BHA. Co-oxidation by prostaglandin H synthase of the BHA-metabolite tert-butylhydroquinone into tert-butylquinone, yielding active oxygen species, might therefore be responsible for the carcinogenic effects of this food antioxidant.

Peroxidation of linoleic, arachidonic and oleic acid in relation to the induction of oxidative DNA damage and cytogenetic effects.

In the present study, the possible role of the polyunsaturated fatty acids linoleic and arachidonic acid in the chemical induction of carcinogenesis has been investigated. Analysis of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG) levels in 2'-deoxyguanosine (dG) and isolated DNA has demonstrated that linoleic and arachidonic acid are capable of inducing this specific genotoxic damage. This effect appears to be related to the degree of fatty acid unsaturation, since it was not induced by monounsaturated oleic acid. Enzymatic peroxidation of linoleic and arachidonic acid resulted in a significant increase in oxidative DNA damage. Studies on the interference of radical scavengers with the induction of 8-oxodG in combination with electron spin resonance spectroscopy demonstrated that the superoxide anion was generated during peroxidation of these fatty acids and that singlet oxygen is most likely involved in the formation of oxidative DNA damage. The level of oxidative damage in dG and single-stranded DNA was higher as compared to that in native DNA after equimolar treatment. Exposure of human lymphocytes to linoleic or arachidonic acid did not result in a significant increase in levels of 8-oxodG. This may indicate that the rate of intracellular peroxidation is relatively low and/or that nuclear DNA in intact cells is effectively protected against genetic damage induced by reactive oxygen species. It is therefore concluded that relatively short periods of linoleic or arachidonic acid administration are not likely to impose a direct genotoxic risk. It can, however, not be excluded that chronic exposure to polyunsaturated fatty acids induces oxidative DNA damage or is related to cancer risk by epigenetic mechanisms, as is also indicated by the observed cytotoxic effects of linoleic and arachidonic acid.

The role of prostaglandin H synthase-mediated metabolism in the induction of oxidative DNA damage by BHA metabolites.

The carcinogenicity of the phenolic food antioxidant butylated hydroxyanisole may be related to its oxidative biotransformation in vivo. In order to determine the ability of BHA, 2-tert-butyl(1,4)hydroquinone (TBHQ) and 2-tert-butyl(1,4)paraquinone (TBQ) to induce oxidative DNA damage, biological inactivation of single-stranded bacteriophage phi X-174 DNA, as well as induction of 7-hydro-8-oxo-2'-deoxyguanosine (8-oxodG) in dG by these compounds was studied in vitro, in the presence and absence of peroxidases. Both test systems showed that BHA and TBQ (probably due to lack of reductase activity in vitro) were not capable of inducting oxidative DNA damage. TBHQ, however, appeared to be a strong inactivator of phage DNA as well as a potent inducer of 8-oxodG formation. Addition of radical scavengers showed that this damage was due to formation of superoxide anion, hydrogen peroxide and hydroxyl radicals. Addition of iron chelators and metal ions showed that the one-electron oxidations of TBHQ via the semiquinone radical into TBQ are toxic via the formation of oxygen radicals and are not directly due to the hydroquinone itself or the formation of semiquinone radicals. Although peroxidation of TBHQ by prostaglandin H synthase (PHS) is indicated to result in a superoxide anion burst, this is not accompanied by an increase in oxidative DNA damage in vitro. This might be due to the use of hydrogen peroxide as a substrate by PHS itself, consequently resulting in less formation of hydroxyl radicals. Oxidation of TBHQ by lipoxygenases showed that no semiquinone radicals or oxygen radicals were formed, probably due to a two-electron oxidation of TBHQ directly into TBQ. The present results indicate that metabolic activation of BHA yielding reactive oxygen species may induce a carcinogenic potential, since the BHA metabolite TBHQ, appeared to be a strong inducer of oxidative DNA damage.