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Archives of Toxicology (v.79, #3)
Role of diacetyl metabolite in alcohol toxicity and addiction via electron transfer and oxidative stress by Peter Kovacic; Andrew L. Cooksy (pp. 123-128).
There are many gaps in our knowledge of the molecular basis of alcohol toxicity and addiction. Metabolism affords mainly acetic acid via acetaldehyde. A minor metabolite, diacetyl (an α-dicarbonyl), arises from the aldehyde. We propose that this C4 entity and/or its iminium derivatives from condensation with protein amino groups plays important roles in bioresponses. A review of the literature reveals substantial support for this premise. Reduction potentials for diacetyl and its iminium derivatives fall in the range favorable for catalytic electron transfer in vivo, which can generate oxidative stress via reactive oxygen species due to redox cycling. Oxidative stress and reactive oxygen species are linked to toxicity associated with major organs by alcohol. The α-dicarbonyl moiety in related substances is believed to induce various toxic responses, such as Alzheimer’s disease, mutagenesis, and carcinogenesis. In addition to discussion of addiction and computational studies, potential applications for health improvement are suggested.
Keywords: Diacetyl metabolite; Alcohol toxicity; Electron transfer; Oxidative stress; Acetaldehyde
Improvements in an in-vitro assay for excitotoxicity by measurement of early gene (c-fos mRNA) levels by Annamarie Rogers; Gabriele Schmuck; Gabriele Scholz; Roger Griffiths; Clive Meredith; Arne Schousboe; Giuseppe Campiani; D. Clive Williams (pp. 129-139).
Quantitative, real-time reverse transcription-polymerase chain reaction (RT-PCR) measurements were made to investigate the levels of c-fos mRNA as one measure of the expression of the c-fos gene. Exposure of mouse cerebellar granule cells to excitotoxic concentrations of glutamate (Glu) and aspartate (Asp) led to a changed time profile for mRNA expression, from a transient c-fos expression at 15–30 min to a delayed, elevated and sustained expression at later time points which was prevented by selective antagonism of the NMDA receptor but not of the AMPA/kainate receptor demonstrating that this c-fos induction was mediated through the specific activation of the NMDA Glu receptor subtype. The question as to whether c-fos expression changes could be used to predict excitotoxicity was addressed by testing the c-fos response of the cultures to several compounds, at low (and therefore non-toxic) and high (toxic) concentrations at two suitable time-points of exposure (30 and 240 min), in the presence and absence of Glu receptor antagonists. The compounds were divided into four groups, excitotoxins, neurotoxic but non-excitotoxic compounds, neuroactive but non-toxic compounds, and compounds that were toxic to other target organelles. The results of this study, using real-time RT-PCR, support the proposal that c-fos mRNA can be used as a specific biomarker of excitotoxicity and moreover encourage further studies to employ this highly sensitive, quantifiable and reproducible technique in a high throughput screen, with minimal use of animals for primary culture set-up. Furthermore, this test has the potential for application in screening newly-designed excitatory amino acid receptor antagonists in the search for clinically relevant drugs to treat a variety of neuropathologies.
Keywords: Excitotoxicityc-fosRT-PCR; In-vitro testing; Glu receptorIegs
Arsenic intoxication-induced reduction of glutathione level and of the activity of related enzymes in rat brain regions: reversal by dl-α-lipoic acid by Samuel Shila; Marimuthu Subathra; Muthuswamy Anusuya Devi; Chinnakkannu Panneerselvam (pp. 140-146).
The purpose of this study was to examine the effects of dl-α-lipoic acid (LA) on arsenic (As) induced alteration of glutathione (GSH) level and of the activity of glutathione-related enzymes—glutathione peroxidase (GSH-Px), glutathione reductase (GR), and glucose-6-phosphate dehydrogenase (G6PDH)—in rat brain regions (cortex, hypothalamus, striatum, cerebellum and hippocampus). Male Wistar rats of 150±10 g weight were divided into four groups: control and three experimental groups supplemented with arsenic (sodium arsenite) alone (100 ppm mixed in drinking water), lipoic acid alone (70 mg kg−1 body weight), arsenic plus lipoic acid (100 ppm arsenic in drinking water plus 70 mg lipoic acid kg−1 body weight). The arsenic content of brain regions was found to increase with the administration of sodium arsenite. Arsenic exposure elicited a significant decline in glutathione content and in the activity of related enzymes, with the greatest decreases seen in the cortex, striatum, and hippocampus, whereas there were no significant differences between control rats and the group treated with lipoic acid alone. Highly elevated content of the thiobarbituric acid-reactive substance malondialdehyde (MDA) in the brain regions of arsenic-exposed rats reflected extensive lipid peroxidation (LPO) processes. Simultaneous lipoic acid treatment was effective in reducing brain regional arsenic levels and lipid peroxidation and in increasing the glutathione content and the activity of its related enzymes. Lipoic acid, by acting as an alternative sulfhydryl nucleophile to glutathione, prevents its oxidation to glutathione disulfide in detoxifying reactions against reactive oxygen species and consequently increases the activity of glutathione-related enzymes.
Keywords: Arsenic; Glutathione; Antioxidant system; Brain regions; Lipoic acid
Species differences in the metabolism of di(2-ethylhexyl) phthalate (DEHP) in several organs of mice, rats, and marmosets by Yuki Ito; Hiroshi Yokota; Ruisheng Wang; Osamu Yamanoshita; Gaku Ichihara; Hailan Wang; Yoshimasa Kurata; Kenji Takagi; Tamie Nakajima (pp. 147-154).
To clarify species differences in the metabolism of di(2-ethylhexyl) phthalate (DEHP) we measured the activity of four DEHP-metabolizing enzymes (lipase, UDP-glucuronyltransferase (UGT), alcohol dehydrogenase (ADH), and aldehyde dehydrogenase (ALDH)) in several organs (the liver, lungs, kidneys, and small intestine) of mice (CD-1), rats (Sprague–Dawley), and marmosets (Callithrix jacchus). Lipase activity, measured by the rate of formation of mono(2-ethylhexyl) phthalate (MEHP) from DEHP, differed by 27- to 357-fold among species; the activity was highest in the small intestines of mice and lowest in the lungs of marmosets. This might be because of the significant differences between Vmax/Km values of lipase for DEHP among the species. UGT activity for MEHP in the liver microsomes was highest in mice, followed by rats and marmosets. These differences, however, were only marginal compared with those for lipase activity. ADH and ALDH activity also differed among species; the activity of the former in the livers of marmosets was 1.6–3.9 times greater than in those of rats or mice; the activity of the latter was higher in rats and marmosets (2–14 times) than in mice. These results were quite different from those for lipase or UGT activity. Because MEHP is considered to be the more potent ligand to peroxisome proliferator-activated receptor α involved in different toxic processes, a possibly major difference in MEHP-formation capacity could be also considered on extrapolation from rodents to humans.
Keywords: Di(2-ethylhexyl) phthalate; Marmoset; Metabolism; Rodent; Species differences
Is the permeability coefficient Kp a reliable tool in percutaneous absorption studies? by Gintautas Korinth; Karl Heinz Schaller; Hans Drexler (pp. 155-159).
In percutaneous absorption studies the potency of penetration of chemical substances is often described by the permeability coefficient Kp. The experimentally determined Kp is characterized according to Fick’s first law of diffusion by the ratio of flux and the concentration of the test compound (Kp=Flux/C). This equation implies that in percutaneous absorption studies Kp is theoretically a more reliable parameter than flux taking the concentration into consideration, and should remain constant for each compound independent from the grade of dilution. In our study we evaluated the course of the percutaneous absorption parameters flux and Kp of neat and of 50% aqueous solution of 2-butoxyethanol (BE). An infinite dose of neat and 50% aqueous solution of non-radiolabeled BE were applied on excised human skin from two donors in static diffusion cells in parallel (for each test setting n=21). The flux of 50% aqueous BE (0.704±0.152 mg/cm2/h) was about 15-fold higher than that of neat BE (0.045±0.014 mg/cm2/h). The comparison of the Kp values of both test settings showed with a factor of about 31 (Kp=1.563×10–3 cm/h) much higher values for 50% aqueous BE and Kp=0.050×10–3 cm/h for neat BE. Although the flux does not consider the chemical concentration, it showed a smaller difference in both test settings as Kp; however, the flux remains a non-specific parameter for the description of percutaneous absorption. The results of our experiments showed that the permeability coefficient Kp was not able to adjust the flux of BE to the concentration. This is in agreement with the evaluation of Kp from BE data described in the literature.
Keywords: Percutaneous absorption; 2-butoxyethanol; Permeability coefficient; Permeability constantKp
Cutaneous metabolism of glycol ethers by David J. Lockley; Douglas Howes; Faith M. Williams (pp. 160-168).
The toxicity of glycol ethers is associated with their oxidation to the corresponding aldehyde and alkoxyacetic acid by cytosolic alcohol dehydrogenase (ADH; EC 1.1.1.1.) and aldehyde dehydrogenase (ALDH; 1.2.1.3). Dermal exposure to these compounds can result in localised or systemic toxicity including skin sensitisation and irritancy, reproductive, developmental and haemotological effects. It has previously been shown that skin has the capacity for local metabolism of applied chemicals. Therefore, there is a requirement to consider metabolism during dermal absorption of these compounds in risk assessment for humans. Cytosolic fractions were prepared from rat liver, and whole and dermatomed skin by differential centrifugation. Rat skin cytosolic fractions were also prepared following multiple dermal exposure to dexamethasone, ethanol or 2-butoxyethanol (2-BE). The rate of ethanol, 2-ethoxyethanol (2-EE), ethylene glycol, 2-phenoxyethanol (2-PE) and 2-BE conversion to alkoxyacetic acid by ADH/ALDH in these fractions was continuously monitored by UV spectrophotometry via the conversion of NAD+ to NADH at 340 nm. Rates of ADH oxidation by rat liver cytosol were greatest for ethanol followed by 2-EE >ethylene glycol >2-PE >2-BE. However, the order of metabolism changed to 2-BE >2-PE >ethylene glycol >2-EE >ethanol using whole and dermatomed rat skin cytosolic fractions, with approximately twice the specific activity in dermatomed skin cytosol relative to whole rat skin. This suggests that ADH and ALDH are localised in the epidermis that constitutes more of the protein in dermatomed skin than whole skin cytosol. Inhibition of ADH oxidation in rat liver cytosol by pyrazole was greatest for ethanol followed by 2-EE >ethylene glycol >2-PE >2-BE, but it only inhibited ethanol metabolism by 40% in skin cytosol. Disulfiram completely inhibited alcohol and glycol ether metabolism in the liver and skin cytosolic fractions. Although ADH1, ADH2 and ADH3 are expressed at the protein level in rat liver, only ADH1 and ADH2 are selectively inhibited by pyrazole and they constitute the predominant isoforms that metabolise short-chain alcohols in preference to intermediate chain-length alcohols. However, ADH1, ADH3 and ADH4 predominate in rat skin, demonstrate different sensitivities to pyrazole, and are responsible for metabolising glycol ethers. ALDH1 is the predominant isoform in rat liver and skin cytosolic fractions that is selectively inhibited by disulfiram and responds to the amount of aldehyde formed by the ADH isoforms expressed in these tissues. Thus, the different affinity of ADH and ALDH for alcohols and glycol ethers of different carbon-chain length may reflect the relative isoform expression in rat liver and skin. Following multiple topical exposure, ethanol metabolism increased the most following ethanol treatment, and 2-BE metabolism increased the most following 2-BE treatment. Ethanol and 2-BE may induce specific ADH and ALDH isoforms that preferentially metabolise short-chain alcohols (i.e. ADH1, ALDH1) and longer chain alcohols (i.e. ADH3, ADH4, ALDH1), respectively. Treatment with a general inducing agent such as dexamethasone enhanced ethanol and 2-BE metabolism suggesting induction of multiple ADH isoforms.
Keywords: 2-Butoxyethanol; 2-Ethoxyethanol; 2-Phenoxyethanol; Cutaneous alcohol dehydrogenase; Hepatic alcohol dehydrogenase; Induction; Multiple exposure
Effects of pesticides on human peripheral lymphocytes in vitro: induction of DNA damage by Ü. Ündeğer; N. Başaran (pp. 169-176).
Because of the widespread use of pesticides for domestic and industrial applications the evaluation of their genotoxic effects is of major concern to public health. Although various experimental data have provided evidence that pesticides can possess genotoxic properties in animals and in in vitro test systems after acute and chronic exposure, the information on the genotoxic effects of some of pesticides is limited and inconsistent. In the present study, the genotoxic potential of commonly used pesticides (i.e., dimethoate and methyl parathion from the organophosphate class, propoxur and pirimicarb from carbamates, and cypermethrin and permethrin from pyrethroids) have been evaluated. The genotoxic effects of these substances were examined using the single cell gel electrophoresis (comet) assay in freshly isolated human peripheral lymphocytes. The cells were incubated with 10, 50, 100 and 200 µg/ml concentrations of the test substances for 0.5 h at 37°C and DNA damage was compared with that obtained in lymphocytes from the same donor not treated with substances. Hydrogen peroxide, 100 µM, was used as a positive control. Within the concentration ranges studied, no significant cytotoxic effects were observed. Dimethoate and methyl parathion at 100 and 200 µg/ml; propoxur at 50, 100 and 200 µg/ml, and pirimicarb, cypermethrin and permethrin at 200 µg/ml significantly increased DNA damage in human lymphocytes.
Keywords: Genotoxicity; Comet assay; Dimethoate; Methyl parathion; Propoxur; Pirimicarb; Cypermethrin; Permethrin
Tumor necrosis factor is not required for particle-induced genotoxicity and pulmonary inflammation by Anne T. Saber; Jette Bornholdt; Marianne Dybdahl; Anoop K. Sharma; Steffen Loft; Ulla Vogel; Håkan Wallin (pp. 177-182).
Particle-induced carcinogenicity is not well understood, but might involve inflammation. The proinflammatory cytokine tumor necrosis factor (TNF) is considered to be an important mediator in inflammation. We investigated its role in particle-induced inflammation and DNA damage in mice with and without TNF signaling. TNF−/− mice and TNF+/+ mice were exposed by inhalation to 20 mg m−3 carbon black (CB), 20 mg m−3 diesel exhaust particles (DEP), or filtered air for 90 min on each of four consecutive days. DEP, but not CB particles, induced infiltration of neutrophilic granulocutes into the lung lining fluid (by the cellular fraction in the bronchoalveolar lavage fluid), and both particle types induced interleukin-6 mRNA in the lung tissue. Surprisingly, TNF−/− mice were intact in these inflammatory responses. There were more DNA strand breaks in the BAL cells of DEP-exposed TNF−/− mice and CB-exposed mice compared with the air-exposed mice. Thus, the CB-induced DNA damage in BAL-cells was independent of neutrophil infiltration. The data indicate that an inflammatory response was not a prerequisite for DNA damage, and TNF was not required for the induction of inflammation by DEP and CB particles.
Keywords: TNF knock-out mice; Lung inflammation; DNA damage; Carbon black; Diesel exhaust particles