Nutraceuticals are widely used by the general public, but very little information is available regarding the effects of nutritional agents on drug toxicity. Excessive doses of acetaminophen (APAP, 4-hydroxyacetanilide) induce hepatic centrilobular necrosis. The naturally occurring substance S-adenosyl-L-methionine (SAMe) has been reported to reduce the hepatic toxicity of APAP. The present study was designed to investigate the hepatoprotective effects of SAMe in comparison to the clinically used antidote N-acetylcysteine (NAC). Male C57BL/6 mice were injected intraperitoneally (i.p.) with an equimolar dose (1.25 mmol/kg) of either SAMe or NAC just before APAP, and the groups were denoted SAMeϩAPAP and NACϩAPAP, respectively. Mice were immediately injected i.p. with 300 mg/kg APAP, and hepatotoxicity was evaluated after 4 h. SAMe was more hepatoprotective than NAC at a dose of 1.25 mmol/kg as liver weight was unchanged by APAP injection in the SAMeϩAPAP group, whereas liver weight was increased in the NACϩAPAP group. SAMe was more hepatoprotective for APAP toxicity than NAC, because alanine aminotransferase levels were lower in the SAMeϩAPAP. Pretreatment with SAMe maintained total hepatic glutathione (GSH) levels higher than NAC pretreatment before APAP, although total hepatic GSH levels were lower in the SAMeϩAPAP and NACϩAPAP groups than the vehicle control values. Oxidative stress was less extensive in the SAMeϩAPAP group compared with the APAPtreated mice as indicated by Western blots for protein carbonyls and 4-hydroxynonenal-adducted proteins. In summary, SAMe reduced APAP toxicity and was more potent than NAC in reducing APAP hepatotoxicity.

Cisplatin, a cancer chemotherapy drug, is nephrotoxic. The aim of this study was to investigate whether resveratrol (RES) reduced cisplatin cytotoxicity and oxidative stress. Rat renal cortical slices were pre-incubated 30 min with 0 (VEH, ethanol) or 30 μg/ml RES followed by 60, 90 or 120 min co-incubation with 0, 75, or 150 μg/mL cisplatin. Lactate dehydrogenase (LDH) leakage was unchanged at 60 and 90 min by cisplatin. Cisplatin increased (p<0.05) LDH leakage at 120 min which was protected by RES. Cisplatin induced oxidative stress prior to LDH leakage as cisplatin depressed glutathione peroxidase and superoxide dismutase (SOD) activity, increased lipid peroxidation, protein carbonyls and 4-hydroxynonenal (4-HNE) adducted proteins within 60 min. RES failed to reverse glutathione (GSH) depression by cisplatin. In order to eliminated an extracellular interaction between RES and cisplatin, additional studies (RINSE studies) allowed a 30 min RES uptake into slices, transfer of slices to buffer lacking RES, followed by 120 min cisplatin incubation. RES in the RINSE studies prevented LDH leakage by cisplatin indicating that RES protection was not via a physical interaction with cisplatin in the media. These findings indicate that RES diminished cisplatin in vitro renal toxicity and prevented the development of oxidative stress.

In the clinical setting, antidotes are generally administered after the occurrence of a drug overdose. Therefore, the most pertinent evaluation of any new agent should model human exposure. This study tested whether acetaminophen (APAP) hepatotoxicity was reversed when S-adenosyl-L-methionine (SAMe) was administered after APAP exposure, similar to what occurs in clinical situations. Comparisons were made for potency between SAMe and N-acetylcysteine (NAC), the current treatment for APAP toxicity. Male C57BL/6 mice were fasted overnight and divided into groups: control (VEH), SAMe treated (SAMe), APAP treated (APAP), N-acetylcysteine treated (NAC), SAMe or NAC administered 1h after APAP (SAMe+APAP) and (NAC+APAP), respectively. Mice were injected intraperitoneal (i.p.) with water (VEH) or 250 mg/kg APAP (15 ml/kg). One hour later, mice were injected (i.p.) with 1.25 mmol/kg SAMe (SAMe+APAP) or NAC (NAC+APAP). Hepatotoxicity was evaluated 4h after APAP or VEH treatment. APAP induced centrilobular necrosis, increased liver weight and alanine transaminase (ALT) levels, depressed total hepatic glutathione (GSH), increased protein carbonyls and 4-hydroxynonenal (4-HNE) adducted proteins. Treatment with SAMe 1h after APAP overdose (SAMe+APAP) was hepatoprotective and was comparable to NAC+APAP. Treatment with SAMe or NAC 1h after APAP was sufficient to return total hepatic glutathione (GSH) to levels comparable to the VEH group. Western blot showed reversal of APAP mediated effects in the SAMe+APAP and NAC+APAP groups. In summary, SAMe was protective when given 1h after APAP and was comparable to NAC.

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Acetaminophen (APAP) hepatotoxicity is protected by S-adenosyl-L-methionine (SAMe) treatment 1 hour (h) after APAP in C57/Bl6 mice. This study examined protein carbonylation as well as mitochondrial and cytosolic protein adduction by 4-hydroxynonenal (4-HNE) using mass spectrometry (MS) analysis. Additional studies investigated the leakage of mitochondrial proteins and 4-HNE adduction of these proteins. Male C57/Bl6 mice (n=5/group) were divided into the following groups and treated as indicated: Veh (15 ml/kg water, ip), SAMe (1.25 mmol/kg, ip), APAP (250 mg/kg), and SAMe given 1 h after APAP (S+A). APAP toxicity was confirmed by an increase (p<0.05) in plasma ALT (U/L) and liver weight/10 g body weight relative to the Veh, SAMe and S+A groups 4 h following APAP treatment. SAMe administered 1 h post APAP partially corrected APAP hepatotoxicity as ALT and liver weight/10 g body weights were lower in the S+A group compared the APAP group. APAP induced leakage of the mitochondrial protein, carbamoyl phosphate synthase-1 (CPS-1) into the cytosol and which was reduced in the S+A group. SAMe further reduced the extent of APAP mediated 4-HNE adduction of CPS-1. MS analysis of hepatic and mitochondrial subcellular fractions identified proteins from APAP treated mice. Site specific 4-HNE adducts were identified on mitochondrial proteins sarcosine dehydrogenase and carbamoyl phosphate synthase-1 (CPS-1). In summary, APAP is associated with 4-HNE adduction of proteins as identified by MS analysis and that CPS-1 leakage was greater in APAP treated mice. SAMe reduced the extent of 4-HNE adduction of proteins as well as leakage of CPS-1.

These data validate the renal cortical slice in vitro model for investigation of radiocontrast nephrotoxicity. These studies further showed that glutathione was cytoprotective. Future research using this model is aimed at further characterization of radiocontrast nephrotoxicity, which may allow for improved prevention and treatment of radiocontrast-induced acute renal failure.

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