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Rat liver microsomal and nuclear activation of methanol to hydroxymethyl free radicals
Gerardo D Castro1, Martín H Costantini, Aurora M A Delgado de Layño
1Centro de Investigaciones Toxicológicas (CEITOX)-CITEFA/CONICET, J.B. de La Salle 4397, B1603ALO Villa Martelli, Buenos Aires, Argentina.
Abstract:
Recent studies from other laboratories reported that during methanol intoxication lipid peroxidation and protein oxidation in liver occurred. Further, they detected free radicals-PBN adducts in bile and urine of methanol poisoned rats. In this work, we report the presence in liver microsomes and nuclei of NADPH dependent processes of hydroxymethyl (HMet) radical formation. The detection of HMet radicals was performed by GC/MS of the trimethylsilyl derivatives of the PBN (N-tert-butyl-a-phenylnitrone)-radical adducts. The formation of HMet radicals was observed only under nitrogen, in these in vitro conditions. Formation of formaldehyde from methanol was observed in aerobic incubation mixtures containing either microsomes or nuclei but also under nitrogen using microsomes. The latter process was not inhibited by diphenyleneiodonium while the anaerobic microsomal one producing HMet was strongly inhibited by it. This shows that they are independent processes. Results suggest that both, liver nuclei and microsomes are able to generate free radicals during NADPH-mediated methanol biotransformation.
Insights
Liver microsomes and nuclei generate hydroxymethyl (HMet) radicals during NADPH-mediated methanol metabolism. These findings reveal new insights into methanol toxicity mechanisms and free radical formation in the liver.
Area of Science:
- Biochemistry
- Toxicology
- Cell Biology
Background:
- Methanol intoxication is linked to liver damage via lipid and protein oxidation.
- Previous studies detected free radicals in bile and urine of methanol-poisoned rats.
Purpose of the Study:
- To investigate NADPH-dependent free radical formation in liver microsomes and nuclei during methanol biotransformation.
- To characterize the hydroxymethyl (HMet) radical and its formation pathways.
Main Methods:
- Detection of HMet radicals using Gas Chromatography/Mass Spectrometry (GC/MS) of PBN (N-tert-butyl-a-phenylnitrone) adducts.
- In vitro incubation of liver microsomes and nuclei with methanol under varying oxygen conditions.
- Inhibition studies using diphenyleneiodonium to differentiate radical formation pathways.
Main Results:
- NADPH-dependent hydroxymethyl (HMet) radical formation was detected in liver microsomes and nuclei under anaerobic conditions.
- Formaldehyde formation from methanol occurred aerobically and anaerobically (microsomes only).
- Anaerobic HMet radical formation in microsomes was sensitive to diphenyleneiodonium, unlike formaldehyde production.
Conclusions:
- Liver nuclei and microsomes possess the capacity to generate free radicals during NADPH-mediated methanol metabolism.
- HMet radical formation and formaldehyde production are distinct, independent processes.
- These findings contribute to understanding the molecular mechanisms of methanol toxicity.