Perturbation of cytochrome P450, generation of oxidative stress and induction of DNA damage in Cyprinus carpio exposed in situ to potable surface water

Epidemiological evidence suggests a link between consumption of chlorinated drinking water and various cancers. Chlorination of water rich in organic chemicals produces carcinogenic organochlorine by-products (OBPs) such as trihalomethanes and haloacetic acids. Since the discovery of the first OBP in the 1970s, there have been several investigations designed to determine the biological effects of single chemicals or small artificial OBP combinations. However, there is still insufficient information regarding the general biological response to these compounds, and further studies are still needed to evaluate their potential genotoxic effects. In the current study, we evaluated the effect of three drinking water disinfectants on the activity of cytochrome P450 (CYP)-linked metabolizing enzymes and on the generation of oxidative stress in the livers of male and female Cyprinus carpio fish (carp). The fish were exposed in situ for up 20 days to surface water obtained from the Trasmene lake in Italy. The water was treated with 1-2 mg/L of either sodium hypochlorite (NaClO) or chlorine dioxide (ClO2) as traditional disinfectants or with a relatively new disinfectant product, peracetic acid (PAA). Micronucleus (MN) frequencies in circulating erythrocytes from the fish were also analysed as a biomarker of genotoxic effect. In the CYP-linked enzyme assays, a significant induction (up to a 57-fold increase in the deethylation of ethoxyresorufin with PAA treatment) and a notable inactivation (up to almost a 90% loss in hydroxylation of p-nitrophenol with all disinfectants, and of testosterome 2 beta-hydroxylation with NaClO) was observed in subcellular liver preparations from exposed fish. Using the electron paramagnetic resonance (EPR) spectroscopy radical-probe technique, we also observed that CYP-modulation was associated with the production of reactive oxygen species (ROS). In addition, we found a significant increase in MN frequency in circulating erythrocytes after 10 days of exposure of fish to water treated with ClO2, while a non-significant six-fold increase in MN frequency was observed with NaClO, but not with PAA. Our data suggest that the use of ClO2 and NaClO to disinfect drinking water could generate harmful OBP mixtures that are able to perturb CYP-mediated reactions, generate oxidative stress and induce genetic damage. These data may provide a mechanistic explanation for epidemiological studies linking consumption of chlorinated drinking water to increased risk of urinary, gastrointestinal and bladder cancers. (c) 2006 Elsevier B.V. All rights reserved

Spectrophotometric method for the determination of polyphenol oxidase activity by coupling of 4-tert-butyl-o-benzoquinone and 4-amino-N,N-diethylaniline

We describe a spectrophotometric analytical method for the detection of polyphenol oxidase activity in an aqueous solution. The assay is based on the coupling reaction between 4-tert-butyl-o-benzoquinone, generated during the enzyme-catalyzed reaction acting on 4-tert-butylcatechol, and the aromatic amine, 4-amino-N,N-diethylaniline to yield a blue adduct (λ(max) 625 nm). This blue adduct exhibited spectral features different from the parent phenol and from the o-quinone. Polyphenol oxidase activities extracted from potatoes, and sunflower seedlings were assayed. The proposed method presents several advantages over the spectrophotometric measurement of 4-tert-butyl-o- benzoquinone. The duration of the linear period was increased, allowing a better determination of its value. The molar extinction coefficient of the blue adduct was higher than that of the 4-tertbutyl-o-benzoquinone; therefore the limitation imposed by the comparatively low e for the 4-tert-butyl-o- benzoquinone is overcome. This is of great importance when considering the inertness of the 4-tert-butyl-o-benzoquinone towards some common coupling agents such as 3-methyl-benzothiazolin-2-one hydrazone

Photometric assay for polyphenol oxidase activity in olives, olive pastes, and virgin olive oils

A photometric method is proposed that allows the determination of phenolase activity in olive fruits, olive pastes, and virgin olive oil. The method can also be used to quantify partially purified phenolase from olives, and is based on the coupling between 4-methyl-o-benzoquinone, the reaction product of phenolase toward its substrate 4-methylcatechol, and the aromatic amine 4-amino-N,N-diethylaniline. The deep-blue adduct arising from this reaction has been characterized by means of nuclear magnetic resonance and mass spectrometric techniques and identified as 4-(4'-diethylamino-phenylimino)-2-hydroxy-5-methyl-cyclohexa-2,5-dienone . This compound shows an absorption band, centered (in dichloromethane) at 617 nm, with an c of 11,080 M(-1)cm(-1). The main advantage of the proposed method resides in the high absorption coefficient of the adduct and its ultraviolet/visible absorption pattern, with a gimel(max) in a spectral region void of significant interferences by the pigments that ultimately will probably be present in the extracts to be tested by this proposed method. The method has proven to be sensitive, specific, and reliable

Insulin secretion defects of human type 2 diabetic islets are corrected in vitro by a new reactive oxygen species scavenger.

Oxidative stress is a putative mechanism leading to beta-cell damage in type 2 diabetes. We studied isolated human pancreatic islets from type 2 diabetic and non-diabetic subjects, matched for age and body mass index. Evidence of increased oxidative stress in diabetic islets was demonstrated by measuring nitrotyrosine concentration and by electron paramagnetic resonance. This was accompanied by reduced glucose-stimulated insulin secretion, as compared to non-diabetic islets (Stimulation Index, SI: 0.9 \ub1 0.2 vs. 2.0\ub10.4, P < 0.01), and by altered expression of insulin (approximately \u201360%), catalase (approximately +90%) and glutathione peroxidase (approximately +140%). When type 2 diabetic islets were preexposed for 24 h to the new antioxidant bis(1-hydroxy-2,2,6,6-tetramethyl-4-piperidinyl)decandioate di-hydrochloride, nitrotyrosine levels, glucose-stimulated insulin secretion (SI: 1.6 \ub1 0.5) and gene expressions improved/normalized. These results support the concept that oxidative stress may play a role in type 2 diabetes beta-cell dysfunction; furthermore, it is proposed that therapy with antioxidants could be an interesting adjunctive pharmacological approach to the treatment of type 2 diabetes

Beneficial effect of the nonpeptidyl low molecular weight radical scavenger IAC on cultured human islet function

We examined a possible protective effect of the nonpeptidyl low molecular weight radical scavenger IAC [bis(l-hydroxy-2,2,6,6-tetramethyl-4-piperidinyl) decanedioate di-hydrochloride] on isolated human islet cells against isolation and culture oxidative stress. Islets isolated from pancreases of nondiabetic multiorgan donors by collagenase digestion were purified by density gradient centrifugation. After the isolation, islets were either exposed or not exposed for 7 days to 10 μmol/L IAC. We found that IAC markedly reduced oxidative stress and ameliorated islets function. These results suggest that the use of IAC could be an interesting pharmacological approach for the treatment of the islets before transplantation. Copyright © 2008 Cognizant Comm. Corp