Genotoxicity of styrene-7,8-oxide and styrene in Fisher 344 rats: a 4-week inhalation study

The cytogenetic alterations in leukocytes and the increased risk for leukemia, lymphoma, or all lymphohematopoietic cancer observed in workers occupationally exposed to styrene have been associated with its hepatic metabolisation into styrene-7,8-oxide, an epoxide which can induce DNA damages. However, it has been observed that styrene-7,8-oxide was also found in the atmosphere of reinforced plastic industries where large amounts of styrene are used. Since the main route of exposure to these compounds is inhalation, in order to gain new insights regarding their systemic genotoxicity, Fisher 344 male rats were exposed in full-body inhalation chambers, 6 h/day, 5 days/week for 4 weeks to styrene-7,8-oxide (25, 50, and 75 ppm) or styrene (75, 300, and 1000 ppm). Then, the induction of micronuclei in circulating reticulocytes and DNA strand breaks in leukocytes using the comet assay was studied at the end of the 3rd and 20th days of exposure. Our results showed that neither styrene nor styrene-7,8-oxide induced a significant increase of the micronucleus frequency in reticulocytes or DNA strand breaks in white blood cells. However, in the presence of the formamidopyridine DNA glycosylase, an enzyme able to recognize and excise DNA at the level of some oxidized DNA bases, a significant increase of DNA damages was observed at the end of the 3rd day of treatment in leukocytes from rats exposed to styrene but not to styrene-7,8-oxide. This experimental design helped to gather new information regarding the systemic genotoxicity of these two chemicals and may be valuable for the risk assessment associated with an occupational exposure to these molecules.     

New scientific arguments for regulation of ethylene oxide residues in skin-care products

Ethylene oxide (EO) occurs as a contaminant of skin-care products because current commercial preparations of polyglycol ethers may contain ethylene oxide monomer residues, up to the order of 1 ppm. Using current regulatory worst-case assumptions, the presence of EO in skin-care products might lead to a maximal human daily external ethylene oxide dose of about 2.8 μg, and a consecutive maximal daily absorbed dose of 0.39 μg. Two methods of toxicokinetic analysis have been used to compare this possible EO load by use of skin-care products with the inevitable load of EO which is produced endogenously in the organism. On the basis of a previous assessment of the endogenous production of ethylene and ethylene oxide (Filser et al. 1992) it is inferred that the absorbed EO dose of 0.39 μg is about 1/30 of the unavoidable human endogenous load by endogenous EO. Alternatively, for a second calculation molecular dosimetry data have been used which were based on experimental quantification of the hydroxyethyl adduct of EO to the N-terminal valine of hemoglobin (HOEtVal) in rats. If the worst-case assumptions for human EO absorption from skin-care products are transferred to the rat species, the associated internal EO doses are about 1/110 of the internal EO doses which were calculated from the background HOEtVal concentrations observed in untreated animals. The divergence between both lines of calculation is mainly due to differences in HOEtVal background concentrations between man and rat. It is concluded that the additional internal body burden of EO associated with the use of current skin-care products, even under a series of worst-case assumptions, is neglegible compared to the physiological and unavoidable internal EO burden of the organism. Received: 21 February 1994/Accepted: 24 March 1994     

Inhalation toxicokinetics of butoxyethanol and its metabolite butoxyacetic acid in the male Sprague-Dawley rat

A total of 16 male Sprague-Dawley rats were continuously exposed to 20 ppm or 100 ppm butoxyethanol (BE) vapor for 1, 2, 3, 4, 6, 8, 10, or 12 days. Urine was collected in 24-h intervals and stored at –70°C. At the end of the exposure the animals were euthanized by decapitation and tissue samples of blood, muscle, liver and were rapidly collected and frozen to –70°C. The samples were later derivatized and analyzed for BE and its major metabolite butoxyacetic acid (BAA) by electron capture gas chromatography. BE and BAA were rapidly distributed to the tissues examined. The concentration of BE in blood was slightly higher, and that of BAA markedly higher than in other tissues, indicating weak (BE) and pronounced (BAA) blood protein binding, respectively. BE was efficiently metabolized and the blood clearance averaged 2.6 l/h per kg, corresponding to a hepatic extraction ratio of about 0.75. The renal clearance of BAA (average 0.53 l/h per kg) corresponded to approximately 15% of the renal blood flow. The kinetics of BE and BAA were linear up to 100 ppm. There were no clear indications of changes in the toxicokinetics, such as metabolic induction or inhibition of metabolism or excretion, during the course of the exposure. The recovery of BAA in urine was 64% of the calculated inhaled amount of BE, on an equimolar basis. Received: 15 February 1994/Accepted: 3 May 1994     

Identification of four metabolites of 3-(phenylamino)alanine,a constituent in l-tryptophan products implicated in eosinophiliamyalgia syndrome,in rats

3-(Phenylamino)alanine (PAA), a contaminant found in L-tryptophan tablets, has been discussed as a possible cause of eosinophilia-myalgia syndrome (EMS). We administered PAA (100 mg/kg) by gastric gavage to Wistar rats to determine its distribution and metabolism. We developed a purification procedure, using Bond Elut SCX cartridges followed by high performance liquid chromatography (HPLC) in order to determine levels of PAA. The level of PAA in blood was 4.22 μg/ml at 5 h and urinary excretion was 21.7 μg for 5 h and 84.6 μg between 5 and 24 h. The amount of PAA in the contents of the large intestine at 5 h was 0.76 μg, indicating poor transfer of PAA to the large intestine. However, the highest concentration of PAA was 12.3 μg/g in the brain, indicating the passage of PAA through the blood-brain barrier. In addition to detecting PAA in the blood and organs, we also detected four metabolites of PAA in urine. We used gas chromatography mass spectrometry to identify PAA in rat liver, as well as N-(hydroxyphenyl)glycine, N-phenylglycine, 3-(pheylamino)lactic acid, and 3-(hydroxyphenylamino)-lactic acid in rat urine. These results suggest that the degradation pathway of PAA is similar to that of phenylalanine. Received: 30 November 1993/Accepted: 25 January 1994     

Effects of deltamethrin on catecholamine secretion of bovine chromaffin cells

The effects of the pyrethroid deltamethrin (D) on catecholamine secretion of cultured bovine chromaffin cells were investigated in vitro using high performance liquid chromatography (HPLC). Spontaneous release of catecholamines was increased by 10 μM and 100 μM D. This increase could partially be prevented by the simultaneous use of 2 μM tetrodotoxin (TTX), which reduced the increase by 10 μM D of catecholamine secretion by 90% and that of 100 μM D by 50%. TTX 2 μM alone did not alter the spontaneous release in comparison to controls. Medullary chromaffin cells consist of two cell groups, one secreting mainly epinephrine (E), the other norepinephrine (NE). The ratio between the spontaneously secreted catecholamines E and NE was increased after treatment with D, indicating a dominant effect on E secreting cells. Received: 7 March 1994/ Accepted: 26 April 1994     

Toxicity of β-blockers in a rat whole embryo culture: concentration-response relationships and tissue concentrations

Beta-adrenoceptor blockers are widely used drugs for the treatment of cardiovascular diseases. Since β-blockers cross the placenta, it is essential to consider possible adverse effects on the embryo. Six β-adrenoceptor blockers were tested at various concentrations (10 – 5000 μM) in a rat whole embryo culture. Although inducing a very similar pattern of dysmorphogenetic effects (incomplete flexure, disturbed development of the neural tube, the head, the heart and the tail bud), the compounds exhibited a wide range of embryotoxic potency. Estimation of the EC₅₀ (median-concentration producing dysmorphogenesis in 50% of the embryos) for the six compounds revealed differences of more than two orders of magnitude: propranolol 25 μM, alprenolol 30 μM, metoprolol 100 μM, pindolol 150 μM, acebutolol 500 μM, atenolol 4000 μM. Measurements of the concentrations of the various drugs in the cultured embryos at corresponding EC₅₀ levels showed differing values: metoprolol 4.5 μM, propranolol 5.2 μM, alprenolol 8.4 μM, pindolol 9.0 μM, acebutolol 12.5 μM and atenolol 77.0 μM. With regard to the EC₅₀ and the degree of substance transfer to the embryo it can be stated that propranolol and metoprolol show a much higher intrinsic potency to interfere with normal in vitro embryonic development than, e.g. atenolol. Received: 1 September 1993 / Accepted: 16 February 1994     

Distribution and reactivity of inhaled 14C-labeled toluene diisocyanate (TDI) in rats

Inhalation exposure to toluene diisocyanate (TDI) can result in a variety of airway diseases. Concern has been expressed that a putative carcinogenic potential of TDI exists as a result of the formation of toluenediamine (TDA) by hydrolysis of the isocyanate in the body. Results from long-term bioassays (TDI inhalation versus gavage in rats and mice) are contradictory and discrepancies do exist concerning the interpretation of adverse effects. This study was performed to analyze the distribution and reactivity of radioactively-labeled TDI using vapor exposure in a rat model system. Rats were exposed to ¹⁴C-TDI vapors at concentrations ranging from 0.026 to 0.821 ppm for 4 h. All tissues examined showed detectable quantities of radioactivity, with the airways, gastrointestinal system and blood having the highest levels which increased with exposure concentration. The concentration of radioactivity in the bloodstream after exposure was linear with respect to dose. The majority (74 – 87%) of the label associated with the blood was recovered in the plasma, and of this, 97 – 100% of the ¹⁴C existed in the form of biomolecular conjugates. Analysis of stomach contents shows that the majority of the label is also associated with high (>10 kDa) molecular weight species. While a larger percentage (28%) of the label is found in the low molecular weight fraction relative to blood, this low molecular weight labeled material represents at least eight different components. Thus, over the vapor exposure concentrations and time tested, it appears that conjugation is the predominant reaction and that free TDA is not a primary in vivo reaction product under the conditions tested. Received: 7 December 1993/Accepted: 7 March 1994     

Induction of single-strand breaks in DNA of mice by trichloroethylene and tetrachloroethylene

Trichloroethylene (TRI) (4-10 mmol/kg body wt) and tetrachloroethylene (PER) (4-8 mmol/kg body wt) were given to male mice by i.p. injection. The induction of single-strand breaks (SSB) in DNA of liver, kidney and lung was studied by the DNA unwinding technique. There was a linear increase of the level of SSB in kidney and liver DNA but not in lung DNA 1 h after administration. The damage was completely repaired 24 h after injection. The capability of TRI and PER to induce SSB in liver DNA is compared to that of three other substances, i.e., methyl methanesulfonate (MMS), styrene-7,8-oxide and styrene, which have been studied earlier by the same technique. The potency of the substances for induction of SSB was in the following order: MMS greater than styrene-7,8-oxide greater than styrene greater than PER greater than TRI.     

Production,characterization and application of monoclonal antibodies against the organophosphorus nerve agent Vx

 Two monoclonal antibodies (Vx-BB8 and Vx-EA11) to the chemical warfare agent Vx were produced and characterized. A competitive inhibition enzyme immunoassay was developed to detect Vx concentrations as low as 3.7×10^-7–3.7×10^-6 mol/l in biological samples. Vx-BB8 400 μg given intravenously immediately before 1×LD₉₅ Vx or 400 μg Vx-BB8 intraperitoneally 1.5 h–3 days before 1×LD₉₅ Vx could protect all the tested mice from death. Received: 14 October 1994/Accepted: 2 February 1995     

Modulation of different stress pathways after styrene and styrene-7,8-oxide exposure in HepG2 cell line and normal human hepatocytes

Styrene is one of the most important monomers produced worldwide. IARC classified styrene as a possible carcinogen to humans (group 2B). Styrene-7,8-oxide (SO) is the main reactive metabolite of styrene, and it is found to be genotoxic in several in vitro test systems.Styrene and styrene-7,8-oxide (SO) toxicity to HepG2 cells was investigated by evaluating end-points such as heat shock proteins (Hsps), metallothioneins (MT), apoptosis-related proteins, accumulation of styrene within the cells and expression of two isoforms of cytochrome P450. The potential activity of styrene and styrene-7,8-oxide in modulating gene expression was also investigated. The results showed induction of Hsp70, metallothioneins, BclX(S/L) and c-myc expression and a decrease in Bax expression in HepG2 after treatments, confirming that these compounds activated protective mechanisms. Moreover, up-regulation of TGFbeta2 and TGFbetaRIII in HepG2 cells was found after exposure to styrene, while in human primary hepatocytes these genes were down-regulated after both treatments. Finally, it was found that styrene and SO treatments did not induce CYP1A2 and CYP2E1 protein expression.In conclusion, both compounds caused toxic stress in HepG2 cells, with SO being more toxic; in the meantime, a different effect of the two compounds in HepG2 cells and primary human hepatocytes was observed regarding their activity in gene modulation.     

Detection of 8-hydroxydeoxyguanosine, a marker of oxidative DNA damage, in white blood cells of workers occupationally exposed to styrene

Styrene-7,8-oxide (SO), the major in vivo metabolite of styrene, is a genotoxic compound and a potential carcinogenic hazard to occupationally exposed workers. The aim of the present work was to investigate the ability of styrene exposure to induce formation of 8-hydroxy-2′-deoxyguanosine (8-OHdG) in white blood cells (WBC) of boatbuilders occupationally exposed to styrene. The study of these adducts was conducted to see if styrene exposure can cause oxidative damage of DNA. The 8-OHdG/10⁵ dG ratio from 17 styrene-exposed workers showed significant increases (mean ± SD, 2.23 ± 0.54, median 2.35, P < 0.001) in comparison to the controls (1.52 ± 0.45, median 1.50). However, 11 out of 17 workers who were between the ages of 32 and 60 years and had been occupationally exposed to styrene for >10 years showed higher 8-OHdG/10⁵ dG ratios (2.31 ± 0.62, median 2.37) in comparison to 6 workers with <6 years of occupational styrene-exposure (2.11 ± 0.36, median 2.05; P > 0.05, no significant difference between the two groups of workers). The studies presented here provide an indication that styrene exposure can result in oxidative DNA damage. Received: 13 January 1997 / Accepted: 24 March 1997     

T(H1)-directed modulation of in vitro cytokine production in human peripheral blood mononuclear cells by styrene-7.8-oxide

Styrene and its chiral main metabolite styrene-7.8-oxide are well characterized regarding their cytotoxic, genotoxic and neurotoxic properties. To our knowledge, no data exist on the influence of styrene and styrene-7.8-oxide on immune reactions. Epidemiological studies suggest that exposure to environmental pollutants, specifically volatile organic compounds (VOCs), including styrene is one factor contributing to increasing prevalence rates of allergic diseases. In this study, we investigated the modulation of the immune system by styrene-7.8-oxide in vitro. Human peripheral blood mononuclear cells were incubated with styrene-7.8-oxide, either as (S)-enantiomer, (R)-enantiomer, or racemic styrene-7.8-oxide. Subsequently, the secretion of T(H1)-cytokines IFNgamma and IL-12 as well as T(H2)-cytokines IL-4 and IL-5 were measured by ELISA. We introduced a novel mathematical approach to quantify and compare cytokine responses. The results revealed a stimulation of cytokine secretion with emphasis on T(H1)-cytokines IFNgamma and IL-12. The stimulating effects were elicited at concentrations of styrene-7.8-oxide comparable to what would be encountered at industrial workplaces where styrene is processed. Therefore, we conclude that styrene-7.8-oxide exhibits immunomodulating capacities, which can be of clinical relevance for individuals with high styrene exposure.     

S-p -Toluylmercapturic acid in the urine of workers exposed to toluene: a new biomarker for toluene exposure

A mercapturic acid attached to the aromatic ring of toluene was for the first time detected in human urine as a metabolite of toluene. Since the metabolism of toluene is usually considered to take place at the side-chain, this gives, besides the biosynthesis of cresols, a further hint of a metabolic conversion of the aromatic system. We examined a group of 33 workers occupationally exposed to toluene, determining the concentrations of toluene in ambient air and in whole blood, o-cresol and hippuric acid in urine and p-toluylmercapturic acid (p-TMA) in urine. All blood and urine samples were collected post-shift. The renal excretion of S-p-toluylmercapturic acid showed highly significant correlations with established parameters of a biological monitoring of toluene. The median ambient air concentration was 63 ppm, ranging from 13 to 151 ppm, the median concentration of toluene in whole blood was 804 μg/l, corresponding to median urinary concentrations for o-cresol of 2.3 mg/l, hippuric acid of 2.3 g/l and p-TMA of 20.4 μg/l. p-TMA was not detectable in urine samples of a control group of 10 non-exposed persons. Both the German Biological Tolerance Values (BAT-values) for toluene in blood (1000 μg/l) and o-cresol in urine (3 mg/l) correspond to a mean p-TMA elimination of ∼50 g/l, and thus are in agreement with each other. According to our results p-TMA reflects internal toluene exposure diagnostically sensitive and specifical. With the developed analytical procedure we determined a median benzylmercapturic acid (BMA) concentration of 190 μg/l in the urine samples of the toluene exposed persons. We also determined a median BMA concentration of 30 μg/l in the control samples of non-exposed persons. However, these results are preliminary and require further confirmation as the reliability of the method was determined only for p-TMA. Received: 15 July 1997 / Accepted: 24 September 1997     

A note on individual differences in the urinary excretion of optical enantiomers of styrene metabolities and of styrene-derived mercapturic acids in humans

 Urine samples from 20 male workers in the polyester industry exposed by inhalation to styrene concentrations ranging from 29 to 41 ppm were investigated. Excretion products of styrene metabolism, mandelic acid and mercapturic acids, were purified from the urine over an extraction column packed with Porapak Q, with subsequent ether elution. The optical enantiomers R- and S-mandelic acid were then determined by thin layer chromatography (TLC) using chiral plate material and selective staining with vanadium pentoxide. Quantitative analysis of these compounds was performed using commercial reference substances. Styrene-specific mercapturic acids were analyzed by a modified TLC method, using synthesized reference substances. The concentration of racemic mandelic acid in the individual urine samples ranged from 80 to 1610 mg/l, and the ratio of the R- and S- enantiomers ranged from 0.7 to 2.2. These individual variations are not explained by differences in individual styrene exposure levels, or by differences in the concentration of the urine samples (in relation to creatinine excretion). Styrene-specific mercapturic acids were detected in the urine of only 1 of the 20 workers, at a concentration much lower than expected from previous investigations by others in humans and laboratory animals, in which less specific analytical methods had been used. The results point to marked interindividual differences in metabolism of styrene, probably related to enzyme polymorphisms. Received: 15 June 1994/Accepted: 3 November 1994     

4-Vinylphenol excretion suggestive of arene oxide formation in workers occupationally exposed to styrene

The toxicity of styrene has often been attributed to the formation of reactive epoxide intermediate, styrene-7,8-oxide. It has been suggested that in addition, an arene oxide, styrene-3,4-oxide, is a metabolite of styrene. Styrene-3,4-oxide is easily converted to corresponding phenols. In this study the presence of 4-vinylphenol in the urine is verified by gas chromatography/mass spectrometry and its quantity compared to mandelic acid excretion. Both 4-vinylphenol and mandelic acid were detected in the urine samples of workers occupationally exposed to styrene. No 4-vinylphenol was found in urine samples of unexposed individuals. The correlation between mandelic acid and 4-vinylphenol was fairly good (r = 0.93); increasing excretion of mandelic acid was also accompanied by increasing amounts of 4-vinylphenol in the urine. The interindividual variation of the 4-vinylphenol/mandelic acid excretion ratio was small, the mean ratio being about 0.3%. The presence of 4-vinylphenol in the urine of workers exposed to styrene suggests that, in man, styrene is also metabolized via arene oxidation. However, when the arene oxidation of styrene is compared to vinyl group oxidation the latter appears to be at least quantitatively by far the more important metabolic pathway.     

Genotoxic risk for humans due to work place exposure to ethylene oxide: remarkable individual differences in susceptibility

Single strand breaks of DNA of peripheral mononuclear blood cells from 97 male and female workers occupationally exposed to ethylene oxide were analysed by the alkaline elution method. These individuals were occupied with the sterilization of medical devices in hospitals and in commercial plants. Ethylene oxide in the air of the working areas was detected up to a maximal concentration of 16.5 mg/m³ calculated as 4-h time-weighted average (4h TWA). Mean value was 1.47±0.52 mg/m³ (1 mg/m³ = 0.55 ppm). Compared to the mean elution rate of the DNA from non-smoking workers exposed to air concentrations of ethylene oxide below the detection limit of 0.1 mg/m³ (4h TWA) the non-smokers working in rooms with a concentration of ethylene oxide between 0.5 mg/m³ and 2 mg/m³ showed a statistically significant (P <0.05) 119% higher mean elution rate and even for the non-smokers exposed to 0.1 – 0.5 mg/m³ of ethylene oxide a statistically significant (P <0.05) 53% higher mean elution rate was observed. For smokers a similar tendency was found but the increase in elution rates in response to the external exposure was smaller than in non-smokers and no statistical significance was obtained. According to their sensitivity to ethylene oxide the non-smoking workers could be classified into two subpopulations. In the majority of the non-smokers (67%) approximately 5-fold more DNA strand breaks were induced by ethylene oxide than in the other non-smokers. A lowest detectable effect level could only be specified for non-smokers. For the “higher sensitive” group the lowest detectable effect level in an examination of a single individual was calculated to be 0.6 mg/m³ ethylene oxide in the air (4h TWA). For the “lower sensitive” group a lowest detectable effect level was calculated to be 3.5 mg/m³. Received: 18 October 1993/Accepted: 16 February 1994     

Pulmonary clearance and inflammatory potency of intratracheally instilled or acutely inhaled nickel sulfate in rats

Rats were exposed to nickel sulfate (NiSO₄) either by intratracheal (IT) instillation or by acute aerosol inhalation, and pulmonary clearance of Ni and pulmonary inflammatory responses were studied. The half-time of Ni in the lung (initial lung burden = 50 μg Ni/rat) was about 32 h in both the IT instillation and inhalation groups. Ni retention in the lung tissue following IT instillation of NiSO₄ was saturable with reference to dose, suggesting that clearance rate of Ni from the rat lung depends on lung burden of Ni. Lung inflammatory responses were evaluated by biochemical, elemental and cytological indicators in bronchoalveolar lavage fluid (BALF) following IT instillation of NiSO₄. Activities of lactate dehydrogenase and β-glucuronidase, contents of lysozyme, protein, sulfur and calcium, and the number of polymorphonuclear leukocytes were increased with a peak at 2 – 3 days post-instillation, while BALF alkaline phosphatase (ALP) activity was significantly decreased after IT instillation of NiSO₄. Lung tissue ALP activity was also decreased by NiSO₄. Because Ni does not inhibit ALP directly, the decrease in ALP activity is probably due to functional changes of type II cells (a major source of BALF ALP). Thiobarbituric acid reacting substances in the lung tissue were not changed by NiSO₄, suggesting that lipid peroxidation plays a minimal, if any role, in the Ni-induced inflammation in the rat lung. Received: 1 December 1993/Accepted: 16 March 1994     

Styrene Inhalation Toxicity Studies in Mice: III. Strain Differences in Susceptibility

Inhalation toxicity studies were conducted to evaluate mousestrain differences in the susceptibility to styrene vapors.Male and female B6C3F1, C57BL/6, Swiss, and DBA/2 mice (8 weeksold) were exposed to 0, 125, 250, or 500 ppm styrene 6 hr/day,for 4 days (20/sex/dose). Histopathological changes and changesin liver weights were evaluated as a measure of hepatotoxicity.Styrene uptake and styrene-7,8-oxide (SO) formation were estimatedby measuring levels of styrene and SO in blood. An estimateof SO detoxification by conjugation with GSH was obtained bymeasuring hepatic GSH depletion. In general, mortality, increasedliver weights, and hepatocellular necrosis were observed inthe 250 and 500 ppm dose groups for all strains and both sexes.Considerable sex and strain differences were observed. Mortality,increased liver weights, and hepatocellular necrosis were greatestin B6C3F1 and C57BL/6 mice in the 250 ppm dose group and inmales; hepatotoxicity was similar in both strains. Swiss miceexhibited dose-dependent increases in mortality, liver weights,and in hepatocellular necrosis, with only slight sex differencesat early time points. Hepatotoxicity in DBA/2, B6C3F1, and C57BL/6strains was greater at 250 than 500 ppm; however, toxicity wasless severe in DBA/2 than in other strains based on absenceof mortality in either sex and less extensive liver necrosisat both 250 and 500 ppm. Blood styrene and SO levels did notcorrelate well with strain differences in toxicity. The relativetoxicity (mortality and hepatotoxicity) was B6C3FI>Swiss>DBA/2;however, relative blood styrene and SO levels were B6C3F1DBA/2>Swiss.Hepatic GSH depletion, (B6C3F1DBA/2>Swiss) correlated withblood SO levels as expected. These results demonstrate significantbiologic variability in the susceptibility of mouse strainsto styrene toxicity. These differences are presumably due tostrain and sex differences in metabolism; however, toxicitydid not correlate well with blood SO levels, suggesting thatother reactive metabolites may contribute to styrene toxicityin mice.     

Influence of glucose on the toxicity of oxophenylarsine in MDCK cells

 Trivalent arsenicals like oxophenylarsine (PhAsO) inhibit cellular pyruvate dehydrogense, thus leading to a drop of acetylCoA formation and a slow-down of the citric acid cycle. Glucose may protect cells from arsenic toxicity, because increased glycolysis may prevent fatal shortage of ATP. On the other hand, PhAsO has been shown to inhibit glucose uptake in Madin-Darby canine kidney (MDCK) cells. We have investigated the effect of PhAsO on viability, ATP levels and glucose uptake of MDCK cells in the presence of normal (5 mmol/l) and low (0.01 mmol/l) glucose concentrations. At normal as well as at low glucose concentrations, cell viability as assessed by formazan formation was not affected by PhAsO concentrations up to 2 μmol/l within 3 h of observation. At higher PhAsO concentrations viability was diminished earlier and more pronounced in the presence of low glucose concentrations. 10 μmol/l PhAsO induced a drastic drop of ATP within 30 min which was followed by an almost complete loss of viable cells after 180 min in the presence of low glucose concentrations, while at normal glucose levels no influence on ATP contents or on cell viability was detected within 60 min of incubation. On the other hand, glucose uptake, determined as ¹⁴C accumulation by cells incubated for 10 min with D[6-¹⁴C]-glucose, was inhibited by PhAsO at low as well as at normal glucose concentrations in a dose dependent manner. At normal glucose concentrations, however, basal uptake was higher (57 nmol/mg protein) and half-maximum inhibition (IC₅₀) was shifted to higher PhAsO concentrations (2×10^-4 mol/l) than at low glucose levels (basal uptake: 1.6 nmol/mg protein; IC₅₀: 5×10^-5 mol/l). It is concluded that 1) in PhAsO-exposed MDCK cells different uptake mechanisms operate in high and low glucose states and 2) that glucose exerts a beneficial effect on the toxicity of PhAsO in these cells. Received: 22 August 1994 / Accepted: 6 December 1994     

Electrophysiological and biochemical effects following single doses of organophosphates in the mouse

Single doses of organophosphates (mipafox or ecothiopate) were given subcutaneously to mice. At intervals up to 77 days after dosing animals were killed and muscle action potentials and endplate potentials were recorded intracellularly in mouse phrenic-nerve/hemidiaphragm preparations. Activities of acetylcholinesterase and neuropathy target esterase in brain and acetylcholinesterase in diaphragm were also measured. Mipafox (0.11 mmol/kg), a neurotoxic organophosphate, produced an increase in prejunctional jitter (i. e. the variabilities of the latencies) of endplate potentials. This increase began 14 – 21 days after administration and lasted more than 23 days. No clinical signs of neuropathy were observed during this study. Mipafox also produced an increase in postjunctional (muscle action potential) jitter. Mipafox inhibited brain and diaphragm acetylcholinesterase and brain neuropathy target esterase. By comparison, a non-neurotoxic organophosphate, ecothiopate (0.5 μmol/kg), was a potent inhibitor of diaphragm acetylcholinesterase and produced a large increase in postjunctional jitter but ecothiopate did not inhibit brain neuropathy target esterase and had no effect on prejunctional jitter. Doses were chosen so that the inhibition of diaphragm acetylcholinesterase by each of the two organophosphates was similar. It is concluded that the neurotoxic organophosphate, mipafox, produced measurable changes in nerve function. These long-term changes may represent a new phenomenon, unrelated to the classical organophosphate induced delayed neuropathy. Alternatively, they may represent a neuropathic process which precedes or is below the threshold for clinical signs. Received: 4 November 1993/Accepted: 2 February 1994