G.A.S. Ansari

High-performance liquid chromatography of cholesterol autoxidation products

Abstract High-performance liquid chromatography with microparticulate silica gel and octadecyl silica reversed-phase columns accords ready resolution of the common autoxidation products of cholesterol. Resolution of two pairs of isomeric sterols (5,6α-epoxy-5α-cholestan-3β-ol and 5,6β-epoxy-5β-cholestan-3β-ol, 3β-hydroxy-5α-cholest-6-ene-5-hydroperoxide and 3β-hydroxycholest-5-ene-7α-hydroperoxide) not previously resolved chromatographically has been achieved in both systems. Application of the procedure to the isolation of cholesta-4,6-dien-3-one from autoxidized cholesterol is described.

Mutagenic cholesterol preparations

Naturally air-aged commercial samples of USP or reagent-grade cholesterol contain components which are mutagenic towards Salmonella typhimurium strains TA1537, TA1538 and TA98. These mutagenic components are associated with the polar cholesterol autoxidation products, but identity of the mutagenic components has not been achieved. Pure crystalline nonmutagenic cholestrol free from autoxidation products becomes mutagenic towards these strains upon heating at 70 degrees in air or following exposure to 60 Co gamma-radiation.

Further investigations of mutagenic cholesterol preparations

The previously demonstrated mutagenicity of naturally air-aged (autoxidized) USP cholesterol in test strains of Salmonella typhimurium has been confirmed. In contrast, autoxidized brassicasterol, 7-dehydrocholesterol, ergosterol, lanosterol, sitosterol, and stigmasterol were non-mutagenic in the same assay, and 38 individual cholesterol derivatives, including many cholesterol autoxidation products, were also non-mutagenic. The mutagenic species from mutagenic cholesterol preparations were shown to be neutral steroids that are very much more polar than the cholesterol autoxidation products so far identified. High-performance liquid chromatography resolved mutagenic cholesterol preparations into several mutagenic and non-mutagenic fractions. The chemical nature of the mutagens is suggested as involving oxidation of the sterol B-ring and of the side-chain.

Fatty acid conjugates of xenobiotics

In this review, we discuss the formation and toxicity of fatty acid conjugates of xenobiotics. Conjugates formed in vivo and in vitro and those detected as contaminants are reviewed. Due to the lipophilic nature of these conjugates, they may accumulate in various body organs and cause toxic manifestations. In vivo formation of these fatty acid conjugates appears to be catalyzed by enzyme(s). Fatty acid ethyl esters are the most widely studied esters and have been implicated in the onset or pathogenesis of myocardial and pancreatic diseases in alcoholics. In experimental animals, studies on 2-chloroethyl linoleate, palmitoylpentachlorophenol and oleoyl and linoleoyl anilides clearly indicate that lipid conjugates of xenobiotics are involved in target organ toxicity. These findings warrant further detailed studies to isolate and identify other fatty acid conjugates and to evaluate their toxicity. Thorough toxicokinetic and metabolic studies are also needed to identify putative toxic agents. Identifying these agents could help in understanding the mechanism of pathogenesis associated with lipid conjugation. Finally fatty acid conjugates of drugs (prodrugs), have been shown to have increased half-lives and long-lasting dose-response. Thus these conjugates may be useful for enhancing the therapeutic potential of drugs and should be explored further.

Chronic exposure to trichloroethene causes early onset of SLE-like disease in female MRL +/+ mice

Trichloroethene (TCE) exacerbates the development of autoimmune responses in autoimmune-prone MRL +/+ mice. Although TCE-mediated autoimmune responses are associated with an increase in serum immunoglobulins and autoantibodies, the underlying mechanism of autoimmunity is not known. To determine the progression of TCE-mediated immunotoxicity, female MRL +/+ mice were chronically exposed to TCE through the drinking water (0.5 mg/ml of TCE) for various periods of time. Serum concentrations of antinuclear antibodies increased after 36 and 48 weeks of TCE exposure. Histopathological analyses showed lymphocyte infiltration in the livers of MRL +/+ mice exposed to TCE for 36 or 48 weeks. Lymphocyte infiltration was also apparent in the pancreas, lungs, and kidneys of mice exposed to TCE for 48 weeks. Immunoglobulin deposits in kidney glomeruli were found after 48 weeks of exposure to TCE. Our results suggest that chronic exposure to TCE promotes inflammation in the liver, pancreas, lungs, and kidneys, which may lead to SLE-like disease in MRL +/+ mice.

In vivo metabolism of the cardiovascular toxin, allylamine

Previous evidence from this laboratory demonstrated that allylamine, a known cardiovascular toxin, is metabolized in vitro to acrolein, which has been hypothesized to act as a distal toxin. In this study, 3-hydroxypropylmercapturic acid was isolated and identified by MS, NMR, and 2D-NMR spectroscopy as the sole urinary metabolite of allylamine metabolism in vivo. Parallel experiments showed reduced glutathione (GSH) depletion in several organs (most marked in aorta, blood, and lung), which is consistent with GSH conjugation of the proposed acrolein intermediate. These findings indicate that allylamine was metabolized in vivo to a highly reactive aldehyde which was converted to a mercapturic acid through a GSH conjugation pathway; the exact mechanisms of cellular damage remain unclear.

THE OXIDATION OF CHOLESTEROL BY HYDROXYL RADICAL

Abstract— The oxidation of chblesterol by hydroxyl radical generated by 60Co y‐radiolysis of aqueous cholesterol dispersions or of dispersions saturated with N2O ave in low yields the products 38‐hydroxy‐cholest‐5‐en‐7‐one. the epimeric cholest‐5‐ene‐3β,7‐diols, 5,6α‐epoxy‐5α‐cholestan‐3β‐ol, 5.6β‐epoxy‐5β‐cholestan‐3,β‐ol, and 5α‐cholestane‐38,5,6β‐triol. Sterol hydroperoxides were not detected except in the radiolysis of dispersions saturated with oxygen.

Increased endobiotic fatty acid methyl esters following exposure to methanol

Human exposure to methanol is likely to increase in the future due to its proposed use as an alternate automobile fuel. Since alcohols are known to esterify the fatty acids in the body and some of those esterified esters are toxic, we studied the formation of fatty acid esters of methanol in Long-Evans male rats given a single oral dose of 3.5 g/kg body weight of methanol in saline. Animals given an equal volume of saline served as control. Three rats were euthanized at 1, 3, 6, 12 and 24 hr following the treatment. Fatty acid methyl esters, extracted from whole blood, liver, pancreas, and brown fat were separated by thin-layer chromatography and quantitated by gas chromatography (GC). Their identity was then confirmed by GC-mass spectrometry. Average levels as high as 596, 5293, 2239, 1106, 9665, 7728, 562, and 2792 micrograms/g (wet weight basis) of 14:0, 16:0, 16:1, 18:0, 18:1, 18:2, 18:3, and 20:4 fatty acid methyl esters, respectively, were found in the pancreas of methanol-treated rats. The average concentration of total fatty acid methyl esters was computed to be 4513, 29594, 22871, 18956, 17014, and 9702 micrograms/g in the pancreas compared to 1.9, 25.4, 36.8, 18.5, 18.9, and 14.2 micrograms/g in the liver at 0, 1, 3, 6, 12, and 24 hr, respectively, following methanol exposure. On dry lipid weight basis, the levels were significantly higher again in pancreas followed by brown fat and liver. In whole blood, only low levels of 16:0, 18:0, and 20:4 fatty acid methyl esters could be detected at all time points. The highest concentration of total fatty acid methyl esters in the pancreas, liver, and brown fat was detected at 1, 3, and 24 hr, respectively. Most of the fatty acid methyl esters found in the liver and pancreas decreased after 6 hr of methanol exposure. The fatty acid methyl esters of higher concentrations were 16:0 in the whole blood, 18:0, 18:1, 18:2, and 20:4 in liver, 18:1, and 18:2 in pancreas and 16:0, 18:1, and 18:2 in brown fat. These fatty acid methyl esters were also detected in the tissues of control rats indicating their endogenous formation. Significant increase in methylation of the fatty acids during methanol exposure, as found in this study, may serve as a defense mechanism for preventing available methanol from oxidative metabolism to render toxicity. However, the biological significance of these fatty acid methyl esters is yet to be understood.

[45] Cholesterol epoxides: Formation and measurement

Publisher Summary This chapter discusses the formation and measurement of cholesterol epoxides. The isomeric cholesterol (1) 5,6-epoxides 5,6α-epoxy-5 α -cholestan-3 β -ol (cholesterol α -oxide, 1) and (2) 5,6 β -epoxy-5 β -cholestan-3 β -ol (cholesterol β -oxide, 2) are found in human tissues and foods and on bioassay exhibit diverse toxic effects in vitro . Both are formed together from cholesterol (cholest-5-en-3 β -ol, 3) by many defined oxidants, including air oxidation, and by the actions of soybean lipoxygenases and of liver microsomal lipid peroxidation systems in vitro . Of all the recognized oxidation products of cholesterol formed by attack on cholesterol of defined active oxygen species, the 5,6-epoxides (1 and 2) are the most broadly formed, being formed by ground state dioxygen ( 3 O 2 ), electronically excited (singlet) dioxygen ( 1 O 2 ), peroxide (O 2 2+ ), ozone (O 3 ), dioxygen cation (O 2+ ), and hydroxyl radical (HO·). The ratio of product 5,6-epoxides (1:2) favors the 5 β ,6 β -epoxide in most cases, the 5 α ,6 α -epoxide predominating only in oxidations involving the specific 5 α ,6 α -epoxidase, HO·, and peracids. In new experimental systems generating the 5,6-epoxides, it is of importance that the products be properly identified and the 5,6-epoxide ratio (1:2) ascertained. The 5α,6α epoxide alone has been posed as the 5,6-epoxide formed in biological systems and as the active agent in numerous bioassays but without adequate supporting evidence.

Selective pancreatic toxicity of palmitoylpentachlorophenol

Palmitoylpentachlorophenol (PPCP), which is a lipid conjugate of a xenobiotic compound, has been found in human fat. To study the toxicity associated with PPCP, rats were given 100 mg/kg PPCP and sacrificed at 4, 8 and 12 days. The target organ identified was the exocrine pancreas; no other major organs examined showed any gross or histopathological abnormality. At 4 and 8 days after treatment, focal, spotty vacuolation, and loss of pancreatic acini was observed. Acute inflammatory infiltrate was also observed in parenchyma at all time points and the loss of acinar tissue was resolved through fibrous tissue formation by 12 days. The present study indicates that PPCP has a specific target organ toxicity.