F. W. Schmahl

Azo dyes and carcinogenic aromatic amines in cell cultures

Abstract Azo dyes are widely used in the food, pharmaceutical, cosmetic, textile and leather industries. They can be reduced by azoreductases in intestinal bacteria, liver cells and skin surface microflora so that aromatic amines are released. In this study an analytical system for the determination of carcinogenic aromatic amines at the picogram to femtogram level and a cell culture assay to evaluate the toxicological effects of azo dyes and aromatic amines is presented. For the assays, the commercial azo dye Resacor Blue 2F (Color Index: Direct Blue 15), a 3,3′-dimethoxybenzidine-based dye, was used. The released carcinogenic aromatic amine, 3,3′-dimethoxybenzidine, was extracted with diethylether derivatized with pentafluoropropionic anhydride and analyzed by capillary gas chromatography/mass spectrometry. In addition, the cytotoxicity of Resacor Blue 2F on cultured kidney epithelial cells and on two hepatocyte cell lines (Hep G2 and Chang liver) was evaluated. For this purpose, the cells were exposed for 72 h to varying concentrations of Resacor Blue 2F. The results show that the azo dye inhibits the proliferation of the kidney epithelial cells much more than the proliferation of the two hepatocyte cell lines. As calculated from the dose-response curves, the EC50 (effective concentration reducing cell proliferation by 50%) for kidney epithelial cells was 40 μg/ml, whereas the EC50 for both hepatocyte cell lines was more than 250 μg/ml. Higher concentrations of 3,3′-dimethoxybenzidine were found in kidney epithelial and in Hep G2 culture supernatants; only small amounts were found in the Chang liver culture supernatant. In summary, it was demonstrated that released 3,3′-dimethoxybenzidine was found in the cell culture supernatants, but it did not accumulate within the cells. For an interpretation of the toxicological results, cell-specific transport systems and osmotic effects of the commercial azo dye which contains several inorganic and organic additives has to be considered.

Nephrotoxic effects of di-(2-ethylhexyl)phthalate (DEHP) hydrolysis products on cultured kidney epithelial cells:

1 Di-(2-ethylhexyl)-phthalate (DEHP) possesses a great industrial value as a plasticizing agent and has become an ubiquitous environmental contaminant. In most species it is rapidly metabolized to mono-(2-ethylhexyl)-phthalate (MEHP) and 2-ethylhexanoic acid (2- EHA). Evaluation of toxicity of DEHP and its primary metabolites has been focussed on reproductive toxicity and hepatocarcinogenic properties. The aim of this study was to determine the nephrotoxic potential of both DEHP metabolites by use of cultured kidney epithelial cells (Opossum kidney cells; OK cells). 2 For this purpose, OK cells were exposed for 3 days to MEHP and 2-EHA at concentrations ranging from 0.1 -500 mmol/L and the toxicity as well as the effects on migratory activity and intracellular cytoskeleton were studied by cell biological, morphological and morpho-metric methods. 3 When compared with corresponding controls, treatment of OK cells with MEHP and 2-EHA, respectively, showed marked differences in cell viability between both DEHP metabolites. MEHP caused a dose-depen-dent decrease in cell viability (ED50 =25 mmol/L) accompanied by a moderate swelling of the cells at concentrations up to 25 mmol/L. MEHP concentrations higher than 25 mmol/L caused a dose-dependent shrinkage of the cells and the occurrence of a high amount of cell debris as a result of cell lysis. 2-EHA did not cause a reduced viability or an altered cell volume. The migratory activity of OK cells was not significantly influenced by both metabolites. Moreover, MEHP toxicity resulted in a largely reduced and altered organization of F-actin (stress fibers), but not of myosin, microtubules and vimentin. 4 The study indicates that cultured epithelial cells can be used as a prescreening system to assess the nephrotoxicity of hazardous substances such as DEHP. As demonstrated in this study, only MEHP, but not 2-EHA, has a marked nephrotoxic effect in vitro.

Stereometabolism of styrene in man: gas chromatographic determination of phenylethyleneglycol enantiomers and phenylethanol isomers in the urine of occupationally-exposed persons.

A gas Chromatographic procedure for the determination of phenylethyleneglycol enantiomers and phenylethanol isomers is described and applied to the investigation of urine samples from occupationally styrene-exposed workers (11 males, six females) and an unexposed control group. Phenylethyleneglycol enantiomers and 2-phenylethanol were present in the urine samples of exposed and unexposed individuals whereas 1-DL-phenylethanol was not found in control urine. The L/D enantiomer ratio of phenylethyleneglycol was found to be approximately 3 in the exposed group and 1.5 in the control group. Because of the close structural relation of these metabolites to the primarily formed epoxide, the results give further insight into the stereotoxicity of styrene in man

Aortic Plaque Size and Endometrial Response in Cholesterol-Fed Rabbits Treated With Estrogen Plus Continuous or Sequential Progestin

ERT is associated with a reduced incidence of coronary risk and cardiac events in postmenopausal women, but increases the risk of endometrial hyperplasia and carcinoma. Combined estrogen and progestin therapy protects the endometrium; however, its effects on heart disease risk factors are not completely known. In our study, 56 ovariectomized New Zealand White rabbits in 7 groups received a 0.5% cholesterol diet for 12 weeks. Controls were not treated with hormones. All other animals received (per kilogram body weight per week) intramuscular injections of either 0.3 mg estrogen (estradiol valerate) alone, 8.3 mg progestin (hydroxyprogesterone caproate) alone, estrogen and progestin continuously in 3 different dosages (0.3 and 8.3 mg; 1 and 8.3 mg; or 1 and 2.8 mg; estrogen and progestin, respectively), or 1 mg estrogen with 25 mg progestin sequentially in 2-week cycles. Eight non-ovariectomized animals served as further controls for endometrial analysis. Morphometric analysis of plaque size in the aortic arch showed that estrogen monotherapy, and the 3 combined therapies with 1 mg estrogen, significantly reduced intimal thickening (P<0.05). The application of progestin alone had no effect on plaque size. The endometrium was enlarged by 3-fold after estrogen treatment, and was decreased by half after progestin treatment, compared with control uteri (P<0.05). In all groups with combined hormone regimens, endometrial size was not significantly different from control uteri. However, these uteri showed more inflammatory reactions, especially when higher doses of hormones were given. In this animal model, doses of progestin that are able to successfully reduce the proliferative effect of estrogen on endometrium do not diminish the desirable antiatherosclerotic properties of estrogen.

Association between method of delivery and puerperal infectious complications in the perinatal database of Baden-Württemberg 1998-2001.

The strongest argument against caesarean delivery relates to maternal complications. Evidence supporting this for elective operations is controversial. The perinatal database 1998–2001 of the German state of Baden-Württemberg was studied to assess the maternal obstetrical risk associated with caesarean delivery with regard to puerperal infectious complications. For statistical analysis the χ2 test, Fisher’s exact test, Mantel-Haenszel statistics and relative risks were used to describe the risk of exposure. Surgical delivery was associated with a significantly higher risk of infectious disorders (p < 0.0001). There was a significantly higher risk of septicaemia in the group undergoing caesarean compared to vaginal delivery (p < 0.0001), for pregnancies with and without risk factors of infection, and also for caesarean delivery prior to labour and rupture of membranes (ROM) and singleton gestations (RR 8.56; 95% CI 4.4–16.65, stratum without risks). The rate of wound disorders was found to be significantly increased in the case of surgical delivery (p < 0.0001). After exclusion of pregnancies with risk factors for infectious complications and multi-fetal gestation, a significantly higher risk was also found for caesarean delivery prior to labour and ROM versus vaginal delivery (RR 16.97; 95% CI 14.16–20.34). Caesarean delivery significantly increased the likelihood that a woman would experience fever in puerperium (p < 0.0001), for pregnancies with and without ante- or perinatal risk factors for infectious complications, and also when caesarean delivery prior to labour and ROM and singletons in the cephalic presentation were considered separately (RR 11.03; 95% CI 9.39–12.96; stratum without risks). Considering the obstetrical challenge of how more women can deliver with fewer complications, reducing unnecessary caesarean delivery still seems to be an appropriate approach.

Post-Prandial Lipaemia after a Moderate Fat Challenge in Normolipidaemic Men with and without Coronary Artery Disease

Background The role of triglycerides as a risk factor for coronary artery disease (CAD) is controversial. In prospective studies, which have reported discrepant results, triglycerides have generally been measured with subjects in the fasting state. Taking into account the fact that the average person spends up to 18 h per day in the post-prandial state, fasting triglyceride levels alone are inadequate to describe the actual effective concentrations, metabolism and atherogenicity of triglyceride-rich lipoproteins. Therefore, investigations of the dynamic post-prandial triglyceride metabolism of patients with CAD in comparison with healthy controls are necessary. Methods We studied the post-prandial metabolism of lipids in 99 men: 50 male patients with CAD confirmed by angiography and 49 matched healthy men. After an overnight fast of 12 h, the subjects (aged 40-60 years) ate a standardized oral fat load (3.2 MJ; 49 g fat = 58% of the total energy content). Blood samples for lipid analyses were drawn immediately prior to and hourly during the 6 h period after ingestion of the meal. Results As required by the study design, fasting triglyceride and total cholesterol levels did not differ between patients and controls; however, high-density lipoprotein (HDL), HDL2 and HDL3 cholesterol levels were significantly lower in the CAD patients. The highest post-prandial triglyceride serum concentrations were observed 3 h after ingestion of the oral fat load both in cases and in controls. Generally, CAD patients had slightly higher triglyceride values than did controls and, at the 5 h measurement point, this difference was significant. Conclusion The results suggest that there are differences in triglyceride metabolism between patients with CAD and healthy controls after a challenge with a moderate amount of fat. These differences can best be observed in the degradation phase of post-prandial lipaemia.

Sodium monochloroacetate causes cytotoxic effects, an increased lactate and pyruvate level and induces ultra structural and cytoskeletal alterations in cultured kidney and liver epithelial cells.

1 Monochloroacetic acid (MCAA) and its sodium salt, sodium monochloroacetate (SMCA) are widely used in chemical industries as intermediates in the synthesis of carboxymethylcellulose, phenoxyacetic acid, thio-glycolic acid, glycine, indigoid dyes and others. More-over, MCAA has been found as a by-product of the chlorination disinfection of drinking water and as an environmental contaminant of the atmosphere from the photodechlorination reactions of chlorinated hydrocarbons. Little is known about the mode of action of both compounds on the cellular level. From cases of accidental poisoning of man it is known that MCAA accumulates in liver and kidney. 2 In this study, the cytotoxicity of SMCA on cultured liver (Chang liver cells) and kidney epithelial cells of the proximal tubule (Opossum kidney cells) was investigated and its effect on metabolism, ultrastruc-ture and organization of cytoskeleton was examined. 3 Independent from the growth state of the cells (proliferating or quiescent), the results clearly show that SMCA causes a dose-dependent decrease in cell viability after an exposure period of 24 h. In all experiments, proliferating cells were more sensitive than quiescent and confluent cells. Liver cells were less sensitive against SMCA treatment than kidney epithe-lial cells. In contrast to liver cells, kidney cells exhibited a dose-dependent decrease in cell volume. The decrease in cell viability was accompanied by an increase of lactate and pyruvate concentrations released into the culture medium. In the case of Opossum kidney cells, lactate and pyruvate levels increased 5-6-fold, whereas in the case of Chang liver cells the increase was approximately twofold. While the ultrastructure of liver cells remained unaltered after drug treatment, kidney cells exhibited cytoplas-mic vacuolization, membraneous disruption and especially mitochondrial alterations. In accordance with the changes in the ultrastructure of Opossum cells, was the reorganization of cytoskeletal elements with an increased stress fiber network at the basolat-eral surface as well as a partial depolymerization of microtubules and vimentin filaments. A cytoskeletal reorganization was not observed for Chang liver cells after SMCA treatment. 4 The results demonstrate that SMCA causes a dose-dependent cytotoxicity which is accompanied by metabolic, mitochondrial and cytoskeletal alterations in the cells.

Stereometabolism of styrene in man

Chiral styrene metabolites obtained during initial styrene exposure of test persons were determined in urine samples using capillary gas chromatography. A typical time-dependent urinary concentration profile of one person over a 49-h period is presented and compared with the results of a previous study of occupationally exposed workers and an unexposed control group. Maximum levels of excretion of all styrene metabolites were observed at about the end of a 9-h workshift. Forty hours after exposure, the L/D-ratio of mandelic acid had subsided to the initial value, and the L/D-ratio of phenylethylene glycol to a value equal or slightly above the initial value.

Cytotoxic effects of 2-butoxyethanol in vitro are related to butoxyacetaldehyde, an intermediate oxidation product

Ethylene glycol ethers belong to a group of solvents with a wide spectrum of applications, particularly because of their compatibility to both hydrophilic and lipophilic systems. Especially ethylene glycol monobutyl ether (2-butoxyethanol, BE) is widely used as a key ingredient in many industrial and consumer cleaning products. Therefore, the risk of human exposure and toxicity by BE as well as its potential for environmental contamination have to be carefully evaluated. By using an established kidney epithelial cell line from the proximal tubule (opossum kidney cells), we investigated the effects of BE on viability, proliferative activity, volume and the organization of the intracellular cytoskeleton of the cells. The experiments were performed with freshly used BE and BE that had been stored at room temperature in the original packing for 3 months after use. After this period of storage the latter BE contained-besides butyraldehyde and n-butanol-0.5 vol% butoxyacetaldehyde (BAL) as measured by capillary gas chromatography and mass spectrometry. Freshly used BE did not cause a toxic effect in the in vitro assays at all concentrations tested (up to 1 mg/ml). In contrast, stored BE which contained BAL reduced cell viability and mitotic activity in a dose-dependent manner. The effective concentration of stored BE causing a 50% loss in cell viability (EC(50/24h)) was calculated to be 1 mg/ml. The toxic effect of stored BE also resulted in alterations of cell morphology and a depolymerization of actin-containing stress fibers. Moreover, administration of stored BE also caused a dose-dependent cell volume increase by the uptake of water, pointing to a necrotic process. In addition, synthesized BAL with a purity of 73.5% (gas chromatography) was also tested and caused an EC(50/24h) of 15 μg/ml, which is a 70-fold lower concentration when compared with stored BE. The present study provides evidence that BE possesses only a low cytotoxic potential in vitro, whereas the corresponding BAL, an intermediate in the oxidation process of BE to butoxyacetic acid, has marked toxic effects. The occurrence of the aldehyde might explain the predominant hematological effects of BE observed in vivo.

New methods for determination of 2-butoxyethanol, butoxyacetaldehyde and butoxyacetic acid in aqueous systems, with special reference to cell culture conditions

Abstract Ethylene glycol ethers, especially 2-ethoxyethanol and 2-butoxyethanol (BE) are frequently used in industry and household as solvents and detergents because of their excellent hydrophilic and lipophilic properties. BE and its oxidation products, butoxyacetaldehyde (BAL) and butoxyacetic acid (BAA), are mainly associated with haemolytic toxicity. No method to determine BAL in aqueous systems (e.g. urine or blood) has been published up to now. BAL was synthesized by dehydration of BE and identified by gas chromatography-mass spectrometry. For determination of BAL and BE with head space-capillary gas chromatography, water and HCl or sodium dihydrogen phosphate were added to the sample. No further extraction or derivatization were necessary. For BAA determination after adding HCl and sodium dihydrogen phosphate the samples were extracted with ethyl acetate and derivatized with 2,2,2-trichloroethanol/HCl. The analytical methods presented here are reliable, sensitive and rapid. The new methods were developed for mammalian cell culture systems, because such in vitro systems are especially useful for metabolic studies and have the advantage of choosing species and organ specificity. In the cell culture experiments presented here it was demonstrated that Opossum kidney cells are able to metabolize BAL to BAA within 24 h. After this interval, in the cells neither BAL nor BAA were accumulated, whereas BAA was found in the cell culture media.