Phthalates rapidly increase production of reactive oxygen species in vivo:Role of Kupffer cells

The role of oxidants in the mechanism of tumor promotion by peroxisome prol iferators remains controversial. The idea that induction of acyl-coenzyme A oxidase leads to increased production of H2O2, which damages DNA, seems un likely; still, free radicals might be important in signaling in specialized cell types such as Kupffer cells, which produce mitogens. Because hard evi dence for increased oxidant production in vivo after treatment with peroxis ome proliferators is lacking, the spin-trapping technique and electron spin resonance spectroscopy were used. Rats were given di(2-ethylhexyl)phthalat e (DEHP) acutely. The spin trapping agent alpha-(4-pyridyl-1-oxide)-N-tert- butylnitrone was also given and bile samples were collected for 4 h. Under these conditions, the intensity of the six-line radical adduct signal incre ased to a maximum value of 2.5-fold 2 h after administration of DEHP, befor e peroxisomal oxidases were induced. Furthermore, DEHP given with [C-13(2)] dimethyl sulfoxide produced a 12-line electron spin resonance spectrum, pro viding evidence that DEHP stimulates (OH)-O-. radical formation in vivo. Fu rthermore, when rats were pretreated with dietary glycine, which inactivate s Kupffer cells, DEHP did not increase radical signals. Moreover, similar t reatments were performed in knockout mice deficient in NADPH oxidase (p47(p hox) subunit). Importantly, DEHP increased oxidant production in wild-type but not in NADPH oxidase-deficient mice. These data provide evidence for th e hypothesis that the molecular source of free radicals induced by peroxiso me proliferators is NADPH oxidase in Kupffer cells. On the contrary, radica l adduct formation was not affected in peroxisome proliferator-activated re ceptor alpha knockout mice. These observations represent the first direct, in vivo evidence that phthalates increase free radicals in liver before per oxisomal oxidases are induced.