Impaired vitamin B12 metabolic status in healthcare workers occupationally exposed to nitrous oxide.

BACKGROUND: Previous studies demonstrated inactivation of vitamin B12 by nitrous oxide (N(2)O). The intraoperative exposure to N(2)O was shown to induce megaloblastic anaemia and myelopathy in subjects with subclinical vitamin B12 deficiency. In contrast, no data concerning the influence of occupational exposure to N(2)O on vitamin B12 metabolic status are available to date. In the present study, the vitamin B12 status in operating theatre personnel was assessed in relation to the extent of exposure. METHODS: Ninety-five operating theatre nurses with the history of exposure to N(2)O and 90 unexposed counterparts were examined. Vitamin B12 and folic acid were measured by immunoassay. Total homocysteine (tHcy), an indicator of impaired vitamin B12 metabolism, was determined by high performance liquid chromatography. N(2)O concentration was monitored by adsorption gas chromatography and mass spectrometry. RESULTS: No significant differences were found between both groups with respect to haematological parameters and folic acid. However, subjects exposed to N(2)O presented with lower vitamin B12 [372.8 (12.1) vs 436.8 (13.2) pmol litre(-1), P<0.001] and higher tHcy [11.2 (0.5) vs 8.9 (0.5) micromol litre(-1), P=0.006]. The changes in vitamin B12 status were aggravated in subjects exposed to N(2)O in concentrations substantially exceeding occupational exposure limit (180 mg m(-3)) [vitamin B12: 341.9 (17.7) vs 436.8 (13.2) pmol litre(-1), P=0.006; tHcy: 12.9 (0.7) vs 8.9 (0.5) micromol litre(-1), P=0.047]. CONCLUSIONS: Exposure to N(2)O in healthcare workers is associated with alterations of vitamin B12 metabolic status, the extent of which depends on the level of exposure.

Increased incidence of micronuclei assessed with the micronucleus assay and the fluorescence in situ hybridization (FISH) technique in peripheral blood lymphocytes of nurses exposed to nitrous oxide.

It has been postulated that exposure to nitrous oxide and halogenated anaesthetics is associated with various adverse health effects such as neurological and reproductive abnormalities or impairment of hepatic functions. In spite of the quite well known genotoxic effects of exposure to nitrous oxide in vivo, the mechanisms of these effects are still not clear. The aim of this study was to assess the frequency of micronuclei and to identify the type of chromosomal damage (clastogenic or aneugenic) in peripheral blood lymphocytes of operating-room nurses exposed to nitrous oxide. The study group comprised 46 women working at departments where the concentration of nitrous oxide ranged from 14 to 2308 mg/m3. The control population was composed of 28 women employed in the same hospitals but in non-surgical departments. The clastogenic/aneugenic effect of nitrous oxide was evaluated in lymphocytes using the standard micronucleus (MN) assay in combination with the fluorescence in situ hybridization (FISH) technique with pancentromeric probes. The results show a significant increase of the MN frequency in lymphocytes of exposed nurses compared with the control group (4.36+/-2.23 versus 9.02+/-4.67). The multiple regression analysis revealed a statistically significant relationship (p=0.0009) between MN frequency and exposure status, indicating that the level of exposure was the main factor affecting chromosomal damage. As assessed by FISH analysis, the overall frequencies of centromere-positive MN in the control and exposed groups were 43 and 49%, respectively. The increase observed in the exposed group may suggest a slight, statistically insignificant pro-aneugenic effect of exposure to nitrous oxide.

Updating of hygiene standards for carbon disulfide based on health risk assessment.

In 1995 the hygiene occupational standard values of carbon disulfide (CS2) were established in Poland: the maximum allowable concentration, eight-hour time weighted average (MAC-TWA)--18 mg/m3, and the short time exposure level (STEL)--30 mg/m3. For lack of reliable retrospective data on the CS2 exposure levels in the work environment and the dose-response relationship, the following have been taken into account in establishing these values: the nervous and vascular systems are recognized as the main CS2 exposure targets; long-term exposure to CS2 in the work environment, exceeding 30 mg/m3, induces the toxic effect in the nervous and cardiovascular systems; chronic exposure to CS2 at concentration below 20 mg/m3 does not produce adverse effects in the peripheral nervous and vascular systems; coronary heart disease does not occur more frequently in workers exposed to CS2. Aiming at updating the 1995 MAC value for CS2 the authors carried out an analysis of the recent literature data on the relation between exposure levels and health risk. The results of clinical and epidemiological studies published in 1995-1997 did not provide evidence that adverse health effects in the cardiovascular and neurological systems in persons occupationally exposed to CS2 at concentration below 48 mg/m3 is likely to occur. The studies of the harmful effects of low CS2 concentration on the reproductive system have not proved that CS2 affects the embryo and fetus. Moreover, in Poland the employment of women under conditions of CS2 exposure (regardless of concentrations) during pregnancy and breast feeding is banned. Because the latest reliable studies have not indicated that chronic CS2 exposure at the level of 20-48 mg/m3 exerts toxic effect on humans, CS2 concentration of 18 mg/m3 as MAC-TWA and 30 mg/m3 as STEL, adopted in 1995, need not to be updated.

Genotoxicity of industrial dyes under the inductive effect of ethanol on monooxygenase system in mice.

The genotoxic effects of triarylmethane (Acid Green 16, C.I.44025) and arylmonoazo (Basic Orange 28, developed by Boruta Pigment Plant, Poland, C.I. undisclosed) dyes, were evaluated in Balb/C mice. Animals were fed for 6 days nutritionally adequate Portagen liquid diet (1 kcal/ml) or isocaloric alcoholic diet containing 5% (w/v) ethanol (36% of total calories) in order to induce the cytochrome P-4502E1 monooxygenase. Dye compounds were administered intraperitoneally 30 h before the test at doses: 90 mg/kg of Acid Green 16 and 70 mg/kg of Basic Orange 28. Bone marrow micronucleus test was used for evaluation of genotoxicity of the dyes. Ethanol caused an increase of the level of cytochrome P-450 by 200% and activities of 7-ethoxycoumarin O-deethylase (ECOD) by 650%, 7-ethoxyresorufin O-deethylase (EROD) by 460% and glutathione (GSH)-S-transferase by 60% in the liver. Both dyes exerted genotoxic effect as inferred from a 3-fold increase of frequency of micronucleated polychromatic erythrocytes in bone marrow, and a further increase (2-fold) was caused by ethanol liquid diet combined with Acid Green 16 treatment. Basic Orange 28 genotoxicity remained unaffected by ethanol. It is concluded that: (1) enhancement of genotoxic effect of Acid Green 16 by ethanol is caused by induction of cytochrome P-4502E1 monooxygenases resulting in an increased bioactivation of the dye; (2) lack of enhancement of the genotoxic effect of Basic Orange 28 by ethanol probably results from the dye- and ethanol-mediated stimulation of GSH-S-transferase, bypassing the cytochrome P-4502E1 bioactivation step.

Carbon disulfide-induced modification and cytotoxicity of low-density lipoproteins.

The secondary oxidation of biologically modified low-density lipoproteins (LDL) was demonstrated to contribute to the cytotoxicity and thereby to the atherogenicity of modified lipoproteins. Previously we have shown that chemical modification of LDL by carbon disulfide (CS(2)) mimicked the naturally occurring process of LDL modification. In the present study the cytotoxicity of CS(2)-modified LDL and their susceptibility to the secondary oxidative modification was investigated. The cytotoxicity of CS(2)-modified LDL did not significantly exceed that of native LDL. However, the Cu(2+) -oxidized form of CS(2)-modified LDL revealed to be more cytotoxic than oxidized native LDL. Oxidized CS(2)-modified LDL presented with altered physicochemical properties including derivatization of protein amino and - SH groups, increased negative charge, and electrophoretic mobility which exceeded that of oxidized native LDL. The secondary oxidative modification of CS(2)-modified LDL involved the process of Cu(2+) binding to CS(2)-derived dithiocarbamate -SH groups followed by covalent modification of - SH groups by products of lipid peroxidation. Taken together, these finding suggest that secondary oxidation of CS(2)-modified LDL may contribute to the atherogenic effect of the chronic occupational exposure to CS(2).

Cytochrome P-450 mediated genotoxicity of triarylmethane dye in mice fed ethanol liquid diet.

Genotoxic effect of synthetic triarylmethane dye (Acid Green 16) was evaluated in Balb C mice fed nutritionally adequate liquid diet (1 kcal/ml) or isocaloric alcoholic diet containing 5% (w/v) ethanol (36% of total calories) for 6 days. Dye compound was given intraperitoneally at dose 150 mg/kg body wt. 30 h before test. The micronucleus test was used for evaluation of genotoxicity of the dye. The level of cytochrome P-450 and the activity of 7-ethoxycoumarin O-deethylase (ECOD) and 7-ethoxyresorufin O-deethylase (EROD) in microsomes were determined to assess the metabolic efficiency of the microsomal system. Acid Green 16 dye provoked an increased frequency of micronucleated polychromatic erythrocytes in bone marrow and ethanol enhanced this genotoxic effect through induction of cytochrome P-4502E1 and stimulation of activities of microsomal monooxygenases (ECOD and EROD), presumably catalyziung bioactivation of the dye.

Urinary excretion of copper bound to low molecular weight proteins in the population exposed to cadmium in community and occupational environments.

People living in Cd-polluted areas excrete increased amounts of copper with urine. A substantial quantity of this is eliminated with metallothionein the concentration of which in urine increases in people exposed to cadmium. Therefore, the measurement of metallothionein in urine is applied as a marker of renal function in people exposed to cadmium in addition to other low molecular weight proteins, beta 2-microglobulins (beta 2MG) and retinol binding proteins (RBP). In this study copper bound to metallothionein-like proteins of low molecular weight (CuBP)--a newly proposed marker of cadmium nephrotoxicity, as well as beta 2MG and RBP, were evaluated in those exposed to cadmium in the community and in occupational environments. The results obtained indicated that people exposed to cadmium in both polluted environments excreted greater amount of CuBP in urine than people not exposed to cadmium. In groups excreting cadmium in urine in amounts ranging from 1 to 10 micrograms/l the urinary level of CuBP was closely associated with the levels of beta 2MG and RBP. A considerable increase in the excretion of urinary CuBP began when Cd concentration in urine exceeded 4 micrograms/l. The amount of CuBP excreted was higher in people with renal disfunction than in those with a normal renal function. It is suggested that urinary excretion of CuBP may be considered as a specific marker of renal function in people exposed to cadmium.

Biological markers of oxidative stress induced by ethanol and iron overload in rat.

Studies on rats treated for 15 months with ethanol (10%, w/v, solution in drinking water) revealed that the stimulation of hepatic cytochrome P-450 monooxygenases activity was accompanied by enhanced microsomal malondialdehyde formation, a lipid peroxidation index and a decreased level of the antioxidant, alpha-tocopherol. The other components of the prooxidant/antioxidant system, diene conjugates and catalase, glutathione peroxidase and superoxide dismutase activities were unaffected. Oxidative stress in blood was shown by a significant decrease in the alpha-tocopherol level whereas lipid peroxidation and antioxidant enzyme activity remained unchanged. The prooxidative effect of ethanol was catalytically promoted by an iron overload (Fe-saccharate, 100 mg Fe3+/kg body wt. intraperitoneally, 2, 5 and 7 day before test) to simulate the effect of alcoholic hemochromatosis. Thus, the level of malondialdehyde and alpha-tocopherol in the serum may be recommended as biological markers of ethanol-provoked oxidative stress, which is especially useful in the evaluation of the combined effect of ethanol and other chemicals that affect the redistribution of active iron complexes.

[Nephrotoxic action of cadmium]. / Nefrotoksyczne dzialanie kadmu.

Functional state of kidney in persons exposed to cadmium (Cd) is estimated mostly by determination of low molecular proteins in urine (beta 2MG, RBP) they are sensitive but not specific indicators of Cd exposure effects. The usefulness of a new indicator of renal functional disorders which is similar to metallothionein was studied. The test involved the determination of the urinary excretion of cadmium bound to low molecular specific protein. It was assessed in population exposed to cadmium in industry (high cadmium concentration) and in population limited to the conditions of residential area (low cadmium concentrations). It was found, that CdBP excretion in urine was higher in a group of persons exposed to cadmium with renal dysfunction (regardless of level of exposure). This was closely correlated with beta 2MG and levels in urine. People with renal dysfunction,-unexposed to cadmium have not excreted any CdBP in urine.

Metabolic disposal of antipyrine and lipid peroxidation indices in volunteers jointly exposed to industrial organic solvents and ethanol.

Each of twelve volunteers, at 2 week intervals, received 1 g of antipyrine, a test drug, and were exposed for 4 h either to toluene (375 mg/m3) or xylene (435 mg/m3) singly or in combination with ethanol (0.45 g/kg body wt. before the onset of exposure and 0.15 g/kg thrice every 1 h during exposure to maintain a steady level of ethanol in blood approximately 11 mmol/dm3). No significant differences were found in salivary antipyrine half-life (T1/2 approximately 12 h); and clearance (ClAP approximately 0.83 cm3/s) between control and groups exposed to solvents and/or ethanol. Nevertheless, a tendency to increase the metabolic rate of antipyrine in xylene-exposed group (T1/2 approximately 6.8 h; ClAP approximately 1.40 cm3/s) and counteraction of ethanol (T1/2 approximately 15 h; ClAP approximately 0.63 cm3/s) should be noted. The stimulation of lipid peroxidation in the serum as a biological effect of combined exposure to ethanol and toluene/xylene was observed.

Genotoxic effect of triarylmethane dye--acid green 16 after chronic ethanol consumption in mice.

Genotoxic effect and hepatic microsomal monooxygenase activities were assessed in mice treated with Acid Green 16 (single i.p. injection at dose 75 mg/kg) superimposed on prolonged ethanol consumption (10% solution in drinking water for 2-4 months). Treatment of mice with Acid Green 16 led to an increased frequency of micronucleated erythrocytes in bone marrow. In animals pretreated with ethanol the frequency of micronucleated erythrocytes, produced by Acid Green 16, was significantly higher. The changes in frequency of micronucleated erythrocytes were accompanied by the enhanced activity of microsomal monooxygenases manifested by higher activity of 7-ethoxycoumarin o-deethylase (the level of cytochrome P-450 was not altered). The obtained results showed that ethanol tended to increase the genotoxic effect of Acid Green 16. However, the slight inductive effect of ethanol on microsomal monooxygenases did not provide clear evidence that the genotoxic effect of Acid Green 16 was associated with ethanol stimulation of the metabolic activation of the dye in the liver.

Interaction of ethanol and xylene in their effects on erythrocytes and other haematological parameters in the rat.

Rats were given ethanol in drinking water for 8 months, followed by inhalation exposure (5 h daily) to 12,000 mg m-3 xylene for 9 days. Combined exposure to xylene and ethanol induced the same changes in the haematological, biochemical and biophysical parameters of the erythrocyte membrane as those found previously in our experiment with toluene-ethanol. Macrocytosis, a decrease in sedimentation rate and erythrocyte packing difference, as well as decreased fluidity of the erythrocytes membrane in the middle zone of the lipid bilayer, were the most significant changes of exposure to ethanol and xylene.

[Induction of cytochrome P-450 monooxygenase after combined exposure of rats to xylene and ethanol]. / Indukcja cytochrom P-450 monooksygenaz pod wplywem kombinowanego narazenia szczurów na ksylen i etanol.

Increased biological risk following from combined exposure to xylene, an industrial solvent, and ethanol, the most likely additional factor to occupational exposure, may result from inductive effects of the chemicals on cyt. P-450 monooxygenase, where biotransformation of xylene and, in part of ethanol, takes place. Studies were carried out on rats: 1) preinduced with ethanol (10% solution in drinking water for 8 months) and for next 9 days jointly exposed to xylene vapour at concentration of 12,000 mg/m3; 2) preexposed to m-xylene vapours at concentration of 4,000 mg/m3 for 6 and 12 weeks or at concentration of 400 mg/m3 for 5 months and for next 3 days jointly administered ethanol (5 doses of 2.5 g/kg in 12 hours intervals). It has been found that ethanol and xylene in both models of combined exposure exert additive stimulatory effects on the activity of cyt. P-450 monooxygenases (aniline p-hydroxylase, microsomal ethanol oxidizing system, NADPH-cyt. c reductase, cytochrome P-450) and intenseness of the effects depends both on the level and duration of exposure to xylene/m-xylene and ethanol.

[Stimulation of microsomal lipid peroxidation as the effect of combined action of xylene and ethanol]. / Stymulacja peroksydacji lipidów mikrosomalnych jako efekt lacznego dzialania ksylenu i etanolu.

The aim of the study was to evaluate if in the case of combined exposure of rats to xylene and ethanol stimulation of lipid peroxidation in the liver microsomes (an index of interaction with lipids and derangements of integrity/fluidity of membranes) might occur. Experiments were carried out on male Wistar rats in the conditions of prolonged, inhalatory preexposure to m-xylene at concentration of 4000 mg/m3 for 6 and 12 weeks, and next joint 5-fold treatment with ethanol (2.5 g/kg oral doses in 12 hours intervals for 3 days). The degree of lipid peroxidation was assessed both in vivo and in vitro under chemical stimulation: enzymatically (NADPH, Fe2+) and nonenzymatically (ascorbic acid, Fe2+). The chemical stimulation permits to measure multiplied biological effects of chemicals acting in vivo. As a results of performed studies it was found that the highest increase of lipid peroxidation appeared in the case of prolonged, 12 weeks exposure to m-xylene (4000 mg/m3) and successively under subacute ethanol treatment and 6-week m-xylene exposure. Thus, it was evidenced that stimulation of lipid peroxidation depends on the duration of exposure to m-xylene. Stimulation of lipid peroxidation, revealed here, may arise from the processes of biotransformation of xylene in cyt. P-450 monooxygenase system where generated oxygen free radicals may attack the lipid components of microsomal membrane as well as from the mechanisms leading to decrease of antioxidant ability of the organism (decrease of glutathione-SH and vitamins E and C levels).

The effect of combined exposures to ethanol and xylene on rat hepatic microsomal monooxygenase activities.

Studies on rats treated for 8 months with ethanol (10% solution in drinking water) and simultaneously exposed to xylene vapour (12,000 mg/m3, 5 hr daily) for the last 9 days revealed that the chemicals exert additive stimulatory effect on hepatic microsomal monooxygenase: the activity of aniline p-hydroxylase increased by 380%, microsomal ethanol oxidizing system by 92%, NADPH-cyt. c reductase by 30% and the level of cytochrome P-450 by 70%. The changes were accompanied by a marked proliferation of smooth endoplasmic reticulum (a subcellular site of cytochrome P-450 monooxygenases in the hepatocytes) and an increased NADPH-Fe2+- and ascorbate-Fe2+-driven lipid peroxidation in microsomal membranes--a potential toxic mechanism. Interaction of ethanol and xylene with cytochrome P-450 monooxygenases may enhance metabolic capacity of the liver and in consequence modify biological/toxic effects of occupational exposure to solvents in the case of alcohol abuse.

Studies on the effect of ethanol and/or toluene on rat erythrocytes.

Rats were subjected to a chronic ethanol exposure in their drinking water for 8 months and then a short subacute toluene exposure to 12,000 mg/m3 for 5 h/day for nine days. Combined exposure increased the reticulocyte count and the concentration of haemoglobin, and changed the biochemical/biophysical properties of red blood cells. Macrocytosis and a decrease in erythrocyte membrane lipid fluidity in the middle zone of the lipid bilayer were the most useful indices of exposure.

Silica earth provoked lung fibrosis with stimulation of lysosomal enzymes and lipid peroxidation in rats.

The dynamics of the biological response of pulmonary tissue to silica dust (silica earth from Piotrowice, Poland, recommended as a domestic reference fibrogenic standard) was studied in rats after single-shot intratracheal instillation of a suspension of 20 mg of the dust for one, three, and seven months. Silica dust provoked pronounced pulmonary fibrosis as inferred from increased collagen content together with pathomorphological alteration (silicotic nodules). The lung burden of silica dust affected the lysosomal subfraction as manifested by an increase in its protein content with concomitant stimulation (release and presumably induction) of beta-glucuronidase and cathepsin D and a transient (up to three months) stimulation of lipid peroxidation. Stimulation of activity of lysosomal enzymes and lipid peroxidation mediated by silica dust may reflect destructive metabolic processes resulting in the development of pulmonary fibrosis as the sign of a pathological repair mechanism. The extent of the effects brought about by silica earth testify that it may be recommended as a reference standard for evaluating the potential health hazard from industrial exposure to dusts containing SiO2.

Combined effect of ethanol and carbon disulphide on cytochrome P-450 mono-oxygenase, lipid peroxidation and ultrastructure of the liver in chronically exposed rats.

A 5-month treatment of rats with ethanol (10% solution in drinking water) stimulated aniline p-hydroxylase and the microsomal ethanol oxidizing system (MEOS) by 140 and 70%, respectively, cytochrome P-450 by 22% and accompanied by lipid peroxidation by 40% in microsomes. It also caused smooth endoplasmic reticulum (SER) proliferation and rough endoplasmic reticulum (RER) degranulation in hepatocytes. Repeated inhalatory exposure of rats to 1.5 g/m3 of CS2, 5 h daily, 5 days a week for 5 months decreased aniline p-hydroxylase and MEOS by 70 and 55% respectively, doubled hexobarbital sleeping time and depressed cytochrome P-450 by 30% and its conversion to cytochrome P-420; these effects were accompanied by the appearance of cytochrome P-420, the intensification of lipid peroxidation in microsomes and some degranulation of RER in hepatocytes. Combined exposure of rats to ethanol and CS2 resulted in a significant potentiation of the inhibitory effects of CS2 on cytochrome P-450 mono-oxygenase and MEOS but with enhancement of CS2 effects on the liver microsomal mono-oxygenase, but CS2 decreased the effect of ethanol on SER proliferation. The interaction both on the biochemical and the morphological level can be explained with the ethanol-stimulated biotransformation of CS2 to reactive electrophilic derivative(s), the subsequent destruction of cytochrome P-450 to cytochrome P-420 and the intensification of lipid peroxidation.

Neurotoxic effects of the combined exposure to carbon disulphide and ethanol in rats.

Neurotoxic effects of the combined exposure of rats to carbon disulphide (CS2) and ethanol (EtOH) were studied. Biochemical and ultrastructural evaluation of the central nervous system (CNS) and peripheral nervous system (PNS) was performed. Male Wistar rats were exposed to CS2 vapour (0.8 mg/l air) and to 10% alcohol in the drinking water for 8 months. EtOH elevated the increase in beta-glucuronidase activity caused by CS2 in the hippocampus and in the cerebral cortex. No effect on the high-affinity synaptosomal uptake of L-glutamate and GABA was observed and no marked ultrastructural changes in the tested brain regions were found. In the peripheral nerves CS2 alone evoked axonal degeneration whereas CS2 combined with EtOH caused disturbances in myelin. Ultrastructural changes preceded biochemical alterations in the PNS and the biochemical indicators of peripheral neuropathy such as beta-glucuronidase activity and cholesterol ester content were not significantly affected. It is suggested that CS2 and EtOH combined affect both PNS and CNS to a higher extent than each of these substances alone.