Carbonyl Reductase Inactivation May Contribute to Mouse Lung Tumor Promotion by Electrophilic Metabolites of Butylated Hydroxytoluene: Protein Alkylation in Vivo and in Vitro

Abstract

Promotion of lung tumors in mice by the food additive butylated hydroxytoluene (BHT) is mediated by electrophilic metabolites produced in the target organ. Identifying the proteins alkylated by these quinone methides (QMs) is a necessary step in understanding the underlying mechanisms. Covalent adducts of the antioxidant enzymes peroxiredoxin 6 and Cu,Zn superoxide dismutase were detected previously in lung cytosols from BALB/c mice injected with BHT, and complimentary in vitro studies demonstrated that QM alkylation causes inactivation and enhances oxidative stress. In the present work, adducts of another protective enzyme, carbonyl reductase (CBR), were detected by Western blotting and mass spectrometry in mitochondria from lungs of mice one day after a single injection of BHT and throughout a 28-day period of weekly injections required to achieve tumor promotion. BHT treatment was accompanied by the accumulation of protein carbonyls in lung cytosol from sustained oxidative stress. Studies in vitro demonstrated that CBR activity in lung homogenates was susceptible to concentration- and time-dependent inhibition by QMs. Recombinant CBR underwent irreversible inhibition during QM exposure, and mass spectrometry was utilized to identify alkylation sites at Cys 51, Lys 17, Lys 189, Lys 201, His 28, and His 204. Except for Lys 17, all of these adducts were eliminated as a cause of enzyme inhibition either by chemical modification (cysteine) or site-directed mutagenesis (lysines and histidines). The data demonstrated that Lys 17 is the critical alkylation target, consistent with the role of this basic residue in NADPH binding. These data support the possibility that CBR inhibition occurs in BHT-treated mice, thereby compromising one pathway for inactivating lipid peroxidation products, particularly 4-oxo-2-nonenal. These data, in concert with previous evidence for the inactivation of antioxidant enzymes, provide a molecular basis to explain lung inflammation leading to tumor promotion in this two-stage model for pulmonary carcinogenesis.

Scientific Opinion on the re‐evaluation of butylated hydroxytoluene BHT (E 321) as a food additive

“…These studies indicate that BHT may exert effects at doses far above the ADI, and some of these effects may be attributed to BHT working as a pro-oxidant at high concentrations, indirectly by inhibiting antioxidant defences through the depletion of nonprotein thiols and by covalent modifications of protective enzymes (Sun et al, 2003). Various mechanisms have been suggested for the occurrence of lung and hepatic tumours in certain strains of mice and rats, and it seems that these effects may be due to many factors, such as inhibition of gap junctional intercellular communication (Guan et al, 1995;Trosko et al, 1990), increase in mitochondrial permeability, epigenetic changes due to induction of DNA methyl transferases (Vanyushin et al, 1998), induction of oxidative stress by the quinone methide metabolites (Faine et al, 2006), inhibition of antioxidant enzymes such as carbonyl reductase (Shearn et al, 2008).…”

Quinone Methides and their Prodrugs: A Subtle Equilibrium Between Cancer Promotion, Prevention, and Cure

“…It decreases calpain-II activity, induces ODC expression (a widely recognized marker of tumor progression) and activates the MAPK family member, ERK, in keratinocytes, thus promoting tumor formation [41,50,51]. BHT also binds and inhibits peroxiredoxin 6, Cu-Zn SOD and CR, which are all antioxidant enzymes [49,52,53].…”

“…The natural products, flavonoids and, especially, quercetin (53), are known for their cancer-preventive properties [56]. Despite these beneficial effects, they also exert toxic effects through transformation into various QMs.…”

Glucuronidation and Methylation of Procyanidin Dimers B2 and 3,3″-Di-O-Galloyl-B2 and Corresponding Monomers Epicatechin and 3-O-Galloyl-Epicatechin in Mouse Liver

“…Combined eluates were concentrated to 0.3 mL under a stream of nitrogen at 25 °C. For analysis of sulfation and methylation, cytosol was prepared from the livers of male C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME) as described (20) and incubated with 0.4 mM substrate at 37 °C for 60 min. Reactions contained 5.0 mg/mL of cytosolic protein and 2.0 mM PAPS (omitted from controls) for sulfation studies or 2.5 mg/mL of protein and 5.0 mM SAM for methylation studies.…”