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Archives of Toxicology (v.77, #7)

Renal cysteine conjugate C-S lyase mediated toxicity of halogenated alkenes in primary cultures of human and rat proximal tubular cells by Trevor A. McGoldrick; Edward A. Lock; Vicente Rodilla; Gabrielle M. Hawksworth (pp. 365-370).

Proximal tubular cells from human (HPT) and rat (RPT) kidneys were isolated, grown to confluence and incubated with S-(1,2-dichlorovinyl)-l-cysteine (DCVC), S-(1,2,2-trichlorovinyl)-l-cysteine (TCVC), S-(1,1,2,2-tetrafluoroethyl)-l-cysteine (TFEC) and S-(2-chloro-1,1-difluorethyl)-l-cysteine (CDFEC), the cysteine conjugates of nephrotoxicants. The cultures were exposed to the conjugates for 12, 24 and 48 h and the toxicity determined using the MTT assay. All four conjugates caused dose-dependent toxicity to RPT cells over the range 50–1,000 μM, the order of toxicity being DCVC>TCVC>TFEC=CDFEC. The inclusion of aminooxyacetic acid (AOAA; 250 μM), an inhibitor of pyridoxal phosphate-dependent enzymes such as C-S lyase, afforded protection, indicating that C-S lyase has a role in the bioactivation of these conjugates. In HPT cultures only DCVC caused significant time- and dose-dependent toxicity. Exposure to DCVC (500 μM) for 48 h decreased cell viability to 7% of control cell values, whereas co-incubation of DCVC (500 μM) with AOAA (250 μM) resulted in cell viability of 71%. Human cultures were also exposed to S-(1,2-dichlorovinyl)-glutathione (DCVG). DCVG was toxic to HPT cells, but the onset of toxicity was delayed compared with the corresponding cysteine conjugate. AOAA afforded almost complete protection from DCVG toxicity. Acivicin (250 μM), an inhibitor of γ-glutamyl transferase (γ-GT), partially protected against DCVG (500 μM)-induced toxicity at 48 h (5% viability and 53% viability in the absence and presence of acivicin, respectively). These results suggest that DCVG requires processing by γ-GT prior to bioactivation by C-S lyase in HPT cells. The activity of C-S lyase, using TFEC as a substrate, and glutamine transaminase K (GTK) was measured in rat and human cells with time in culture. C-S lyase activity in RPT and HPT cells decreased to approximately 30% of fresh cell values by the time the cells reached confluence (120 h), whereas the decline in GTK activity was less marked (50% of the fresh cell values at confluence). Rat cells had threefold higher activity than human cells at each time point. This higher activity may partly explain the differences in toxicity between rat and human proximal tubular cells in culture.

Keywords: C-S lyase; Halogenated alkenes; Human proximal tubular cells; Rat proximal tubular cells

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) alters the endogenous metabolism of all-trans-retinoic acid in the rat by Carsten K. Schmidt; Pi Hoegberg; Nicholas Fletcher; Charlotte B. Nilsson; Christina Trossvik; Helen Håkansson; Heinz Nau (pp. 371-383).

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is known to influence vitamin A homeostasis. In order to investigate the mechanism behind this retinoid disruption, male Sprague-Dawley rats were exposed to TCDD at doses ranging from 0.1 to 100 µg/kg body weight, and were killed 3 days after exposure. Additional groups of rats were killed 1 and 28 days after a single oral dose of 10 µg TCDD/kg body weight. Serum, kidney, and liver were investigated for retinoid levels, as well as gene expression and enzyme activities relevant for retinoid metabolism. Besides the well known effects of TCDD on apolar retinoids, i.e. decreased hepatic and increased renal retinyl ester (RE) levels, we have found dose-dependent elevation of all-trans-retinoic acid (all-trans-RA) levels in all investigated tissues. In the liver, 9-cis-4-oxo-13,14-dihydro-RA was drastically decreased by TCDD in a dose-dependent manner. In serum, cis-isomers of all-trans-RA, including 9,13-di-cis-RA, were significantly reduced already at the lowest dose level. Protein and mRNA levels of cellular retinol binding protein I (CRBP-I) in liver or kidneys were not significantly altered by TCDD exposure at doses at which retinoid levels were affected, making CRBP-I an unlikely candidate to account for the alterations in retinoid metabolism caused by TCDD. The expression and activities of relevant cytochrome P450 (CYP) enzymes with potential roles in all-trans-RA synthesis and/or degradation (CYP1A1, 1A2, and 2B1/2) were also monitored. A possible role of CYP1A1 in TCDD-induced all-trans-RA synthesis is suggested from the time-course relationship between CYP1A1 activity and all-trans-RA levels in liver and kidney. The significant alteration of the all-trans-RA metabolism has the potential to contribute significantly to the toxicity of TCDD.

Keywords: TCDD; Vitamin A; All-trans-retinoic acid; 9,13-Di-cis-retinoic acid; Metabolism; 9-Cis-4-oxo-13,14-dihydro-retinoic acid; Cellular retinol binding protein I; Cytochrome P450; Rat

The cytotoxicity of jet fuel aromatic hydrocarbons and dose-related interleukin-8 release from human epidermal keratinocytes by C. C. Chou; J. E. Riviere; N. A. Monteiro-Riviere (pp. 384-391).

Many jet fuel aromatic hydrocarbons are known carcinogens with the ability to both readily penetrate the skin with high absorptive flux and cause skin irritation. In order to evaluate the in vitro cutaneous toxicity of individual aromatic hydrocarbons in jet fuels and their potential for inducing skin irritation, we evaluated the LD₅₀, the highest non-cytotoxic (5% mortality) dose (HNTD), and interleukin-8 (IL-8) release activity of nine major jet fuel aromatic hydrocarbons in human epidermal keratinocytes (HEK). LD₅₀ ranged from 1.8 mM (0.03%) for cyclohexylbenzene to 82.9 mM (0.74%) for benzene, with a rank order potency of cyclohexylbenzene >trimethylbenzene ≥xylene >dimethylnaphthalene >ethylbenzene >toluene >benzene. The HNTD values ranged from 0.1 mM (0.001%) for cyclohexylbenzene to 48.2 mM (0.43%) for benzene. Naphthalene and methylnaphthalene could not be ranked in this comparison since their concentrations, presented as percentage saturation, were not comparable to the others presented as solutes in solution. There was a dose-related differential response in IL-8 release at 24 h. Toluene, xylene, trimethylbenzene, cyclohexylbenzene and dimethylnaphthalene significantly decreased IL-8 release at the respective HNTDs, while IL-8 release did not continue to decrease, or significantly increased (cyclohexylbenzene and dimethylnaphthalene), at the LD₅₀. IL-8 significantly increased with both doses of methylnaphthalene and naphthalene. The presence of hexadecane and mineral oil greatly attenuated the cytotoxicity elicited by individual aromatic hydrocarbons in HEK cells.

Keywords: Aromatic hydrocarbons; Cytotoxicity; Keratinocytes; Interleukin-8; Jet fuels

Markers of experimental acute inflammation in the Wistar Han rat with particular reference to haptoglobin and C-reactive protein by P. S. Giffen; J. Turton; C. M. Andrews; P. Barrett; C. J. Clarke; K.-W. Fung; M. R. Munday; I. F. Roman; R. Smyth; K. Walshe; M. J. York (pp. 392-402).

C-reactive protein (CRP), haptoglobin (Hp) and fibrinogen (Fbgn) are acute phase reactants (APRs), the blood levels of which increase during acute inflammation. However, although the levels of these APRs are used to monitor inflammation in man, their usefulness and sensitivity as markers of inflammation in rodents are less clear. We therefore wished to evaluate, in a comparative fashion, a prototype immunoassay for serum CRP, a commercial assay for serum Hp, and an automated assay for Fbgn, using a model of acute inflammation in the rat. Additionally, pro-inflammatory cytokines and serum protein fractions were also measured. The model of inflammation used was the intraperitoneal injection of Freund's complete adjuvant (FCA). In a concluding experiment, findings with Hp in the FCA rat model were validated in a toxicologically relevant study involving the induction of acute hepatic inflammation using the model hepatotoxicant carbon tetrachloride (CCl₄). Female Wistar Han rats were treated with a single injection of FCA in a dose–response study (1.25–10.0 ml/kg, sampling at 36 h) and two time-course studies (over 40 h and 21 days). In a final experiment, rats were dosed with CCl₄ at 0.8 ml/kg and sampled over a 17-day period. In FCA and CCl₄ experiments, serum/plasma was prepared and tissues taken at autopsy for histological assessment (CCl₄ study only). In the dose–response study, serum CRP, Hp and plasma Fbgn were increased at all FCA dose levels at 36 h post-dosing. Serum α₂ and β₁ globulin fractions were also increased, while albumin levels were decreased. In the 40-h time-course study, CRP levels peaked at 25–40 h post-dosing, to approximately 120% of control (as 100%). Hp levels increased to a maximum at 25 and 40 h post-dosing with values greater than 400% of control, and α₂ and β₁ globulin fractions peaked at 30 and 40 h post-dosing to 221 and 187% of control, respectively. Increased serum interleukin-6 (IL-6) and interleukin-1β (IL-1β) levels peaked at 20 h (11-fold) and 25 h (19-fold), respectively. In a 21-day time-course study, no increased CRP levels were measured despite elevated levels of Hp, which peaked at 36 h (approximately 7-fold above control), and remained elevated up to 21 days. IL-6 and IL-1β levels peaked at 12 h (19-fold) and 24 h (28-fold), respectively. Liver histopathology of animals treated with CCl₄ showed centrilobular hepatocellular degeneration and necrosis (most significant at 36 h) with an inflammatory response (most significant at 48 h). Resolution of the lesion was complete by 4 days post-dosing. Serum alanine aminotransferase, aspartate aminotransferase and glutamate dehydrogenase levels peaked at 36 h post-dosing. Hp levels increased maximally at 48 h (426% of control). We conclude that serum CRP is a poor marker of acute inflammation in the rat in comparison with serum Hp and plasma Fbgn. Between Hp and Fbgn, serum Hp is shown to be the most sensitive and useful marker of acute inflammation.

Keywords: Acute phase reactant; Freund's complete adjuvant; C-reactive protein; Fibrinogen; Haptoglobin; Inflammation; Carbon tetrachloride

The herbicide dicamba (2-methoxy-3,6-dichlorobenzoic acid) interacts with mitochondrial bioenergetic functions by Francisco Peixoto; Joaquim A. F. Vicente; Vítor M. C. Madeira (pp. 403-409).

The effects of dicamba, a widely used broad-leaf herbicide, on rat liver mitochondrial bioenergetic activities were examined. The results obtained for state 4 respiration indicate not only an uncoupling effect, the result of an increase on the permeability of inner mitochondria membrane to protons, but also a strong inhibitory effect on the redox complexes. State 3 and respiration uncoupled by FCCP (carbonylcyanide p-trifluoromethoxyphenylhydrazone) were inhibited to approximately the same extent, i.e. by about 70%. Depression of respiratory activity is essentially mediated through partial inhibition of mitochondrial complexes II and III. ATPase activity was much less depressed by dicamba than ATP synthase activity. Therefore, a considerable part of the inhibition observed on ATP synthase is the result of an inhibition on the redox complexes. The loss of phosphorylation capacity, induced by dicamba, was in the last analysis not only the result of a direct effect of dicamba on the enzymatic complex (F₀–F₁ ATPase) but also the result of a deleterious effect on the integrity of the mitochondrial membrane, which can promote an inhibition of the respiratory complexes and an increase of the proton permeability of the inner membrane.

Keywords: Herbicide; Dicamba; Mitochondria; Oxidative phosphorylation

Hexose monophosphate shunt activities in human erythrocytes during oxidative damage induced by hydrogen peroxide by J. Guitton; S. Servanin; A. Francina (pp. 410-417).

Human red blood cells (HRBCs) were exposed to H₂O₂ either as bolus or as a flux generated by a glucose–glucose oxidase system. H₂O₂ concentrations were in the range 10⁻⁵–10⁻³ M and exposure times to the oxidative stress were 10 min and 60 min. The production of NADPH by the hexose monophosphate shunt (HMPS) was accurately measured by gas chromatography–isotope ratio mass spectrometry as the production of ¹³CO₂ from [1-¹³C]glucose. Depending on the duration of exposure and H₂O₂ concentration, the production of ¹³CO₂ by HRBCs under a flux of H₂O₂ was increased two- to eight-fold in comparison with that obtained under a bolus of H₂O₂. Under flux stimulation, spectral data show the formation of compound I, and a red shift caused by the presence of compounds II and III, whereas under a bolus stress no obvious spectra changes were observed. Inhibition of catalase by 3-amino-1,2,4-triazole (3-AT) or by sodium azide, followed by a bolus of H₂O₂ led to a two- to five-fold increases in ¹³CO₂ production compared with controls, depending on H₂O₂ concentration. In contrast, 3-AT-inhibited HRBCs exposed to a flux of H₂O₂ did not present an increase in ¹³CO₂ production. The present paper emphasizes the importance and role of NADPH production following a bolus or a flux stimulation of H₂O₂. The difference between responses in HMPS activities under the two types of stress could be related to a different balance of activity between 'catalatic' and 'peroxidatic' modes of catalase following H₂O₂ exposure.

Keywords: Catalase; NADPH; Erythrocyte; Hydrogen peroxide; Gas chromatography–isotope ratio mass spectrometry

Propofol reduces nitric oxide biosynthesis in lipopolysaccharide-activated macrophages by downregulating the expression of inducible nitric oxide synthase by Ruei-Ming Chen; Gong-Jhe Wu; Yi-Ting Tai; Wei-Zen Sun; Yi-Ling Lin; Wen-Chi Jean; Ta-Liang Chen (pp. 418-423).

Nitric oxide is an active oxidant that contributes to the physiology and pathophysiology of macrophages. Propofol has been widely used in intravenous anesthesia. It possess antioxidant and immunomodulating effects. This study aimed to evaluate the effects of propofol on nitric oxide production in lipopolysaccharide-activated macrophages. Exposure of macrophages to propofol (25, 50 and 75 µM), to lipopolysaccharide (0.5, 1, 1.5 and 2 ng/ml) or to a combination of propofol and lipopolysaccharide did not affect cell viability. However, propofol at 100 µM led to significant cell death (P<0.05). The levels of nitrite, an oxidative product of nitric oxide, were increased in lipopolysaccharide-treated macrophages in a concentration-dependent manner (P<0.01), while propofol could concentration-dependently decrease the lipopolysaccharide-enhanced nitrite levels (P<0.01). Immunoblotting analysis revealed that lipopolysaccharide increased the protein level of inducible nitric oxide synthase (iNOS). The co-treatment of propofol and lipopolysaccharide significantly reduced this lipopolysaccharide-induced iNOS protein (357±49×10³ versus 92±6×10³ arbitrary units, P<0.01). Analysis by reverse transcriptase-polymerase chain reaction showed that lipopolysaccharide induced mRNA of iNOS, but that the inductive effect was inhibited by propofol (95±7×10² versus 30±4×10² arbitrary units, P<0.01). This study has demonstrated that propofol, at therapeutic concentrations, could suppress nitric oxide biosynthesis by inhibiting iNOS expression in lipopolysaccharide-activated macrophages. The mechanism of suppression was at a pretranslational level.

Keywords: Propofol; Macrophages; Lipopolysaccharide; Inducible nitric oxide synthase; Nitric oxide

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Archives of Toxicology (v.77, #7)