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 proliferators remains controversial. The idea that induction of acyl-coenzyme A oxidase leads to increased production of H₂O₂, which damages DNA, seems unlikely; still, free radicals might be important in signaling in specialized cell types such as Kupffer cells, which produce mitogens. Because hard evidence for increased oxidant production in vivo after treatment with peroxisome proliferators is lacking, the spin-trapping technique and electron spin resonance spectroscopy were used. Rats were given di(2-ethylhexyl) phthalate (DEHP) acutely. The spin trapping agent α-(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 increased to a maximum value of 2.5-fold 2 h after administration of DEHP, before peroxisomal oxidases were induced. Furthermore, DEHP given with [¹³C₂]dimethyl sulfoxide produced a 12-line electron spin resonance spectrum, providing evidence that DEHP stimulates OH radical formation in vivo. Furthermore, when rats were pretreated with dietary glycine, which inactivates Kupffer cells, DEHP did not increase radical signals. Moreover, similar treatments were performed in knockout mice deficient in NADPH oxidase (p47^phox subunit), importantly, DEHP increased oxidant production in wild-type but not in NADPH oxidase-deficient mice. These data provide evidence for the hypothesis that the molecular source of free radicals induced by peroxisome proliferators is NADPH oxidase in Kupffer cells. On the contrary, radical adduct formation was not affected in peroxisome proliferator-activated receptor α knockout mice. These observations represent the first direct, in vivo evidence that phthalates increase free radicals in liver before peroxisomal oxidases are induced.