Chemoselective and Highly Sensitive Quantification of Gut Microbiome and Human Metabolites.

The microbiome has a fundamental impact on the human host's physiology through the production of highly reactive compounds that can lead to disease development. One class of such compounds are carbonyl-containing metabolites, which are involved in diverse biochemical processes. Mass spectrometry is the method of choice for analysis of metabolites but carbonyls are analytically challenging. Herein, we have developed a new chemical biology tool using chemoselective modification to overcome analytical limitations. Two isotopic probes allow for the simultaneous and semi-quantitative analysis at the femtomole level as well as qualitative analysis at attomole quantities that allows for detection of more than 200 metabolites in human fecal, urine and plasma samples. This comprehensive mass spectrometric analysis enhances the scope of metabolomics-driven biomarker discovery. We anticipate that our chemical biology tool will be of general use in metabolomics analysis to obtain a better understanding of microbial interactions with the human host and disease development.

Electronic cigarette solvents, pulmonary irritation, and endothelial dysfunction: role of acetaldehyde and formaldehyde.

After more than a decade of electronic cigarette (E-cig) use in the United States, uncertainty persists regarding E-cig use and long-term cardiopulmonary disease risk. As all E-cigs use propylene glycol and vegetable glycerin (PG-VG) and generate abundant saturated aldehydes, mice were exposed by inhalation to PG-VG-derived aerosol, formaldehyde (FA), acetaldehyde (AA), or filtered air. Biomarkers of exposure and cardiopulmonary injury were monitored by mass spectrometry (urine metabolites), radiotelemetry (respiratory reflexes), isometric myography (aorta), and flow cytometry (blood markers). Acute PG-VG exposure significantly affected multiple biomarkers including pulmonary reflex (decreased respiratory rate, -50%), endothelium-dependent relaxation (-61.8 ± 4.2%), decreased WBC (-47 ± 7%), and, increased RBC (+6 ± 1%) and hemoglobin (+4 ± 1%) versus air control group. Notably, FA exposure recapitulated the prominent effects of PG-VG aerosol on pulmonary irritant reflex and endothelial dysfunction, whereas AA exposure did not. To attempt to link PG-VG exposure with FA or AA exposure, urinary formate and acetate levels were measured by GC-MS. Although neither FA nor AA exposure altered excretion of their primary metabolite, formate or acetate, respectively, compared with air-exposed controls, PG-VG aerosol exposure significantly increased post-exposure urinary acetate but not formate. These data suggest that E-cig use may increase cardiopulmonary disease risk independent of the presence of nicotine and/or flavorings. This study indicates that FA levels in tobacco product-derived aerosols should be regulated to levels that do not induce biomarkers of cardiopulmonary harm. There remains a need for reliable biomarkers of exposure to inhaled FA and AA.NEW & NOTEWORTHY Use of electronic cigarettes (E-cig) induces endothelial dysfunction (ED) in healthy humans, yet the specific constituents in E-cig aerosols that contribute to ED are unknown. Our study implicates formaldehyde that is formed in heating of E-cig solvents (propylene glycol, PG; vegetable glycerin, VG). Exposure to formaldehyde or PG-VG-derived aerosol alone stimulated ED in female mice. As ED was independent of nicotine and flavorants, these data reflect a "universal flaw" of E-cigs that use PG-VG.Listen to this article's corresponding podcast at https://ajpheart.podbean.com/e/e-cigarettes-aldehydes-and-endothelial-dysfunction/.

Detection of Volatile Organic Compounds in a Drop of Urine by Ultrasonic Nebulization Extraction Proton Transfer Reaction Mass Spectrometry.

Detection of volatile organic compounds (VOCs) in human urine has potential application value in screening for disease and toxin exposure. However, the current technologies are too slow to detect the concentration of VOCs in fresh urine. In this study, we developed a novel ultrasonic nebulization extraction proton transfer reaction mass spectrometry (UNE-PTR-MS) technology. The urinary VOCs can be rapidly extracted to gaseous VOCs using the UNE system and then delivered using a carrier gas to the PTR-MS instrument for rapid detection. The carrier gas flow and sample size were optimized to 100 mL/min and 100 µL, respectively. The limits of detection (LODs) and response time of the UNE-PTR-MS were evaluated by detecting three VOCs that are common in human urine: methanol, acetaldehyde, and acetone. The LODs determined for methanol (4.47 µg/L), acetaldehyde (1.98 µg/L), and acetone (3.47 µg/L) are 2-3 orders of magnitude lower than the mean concentrations of that in healthy human urine. The response time of the UNE-PTR-MS is 34 s and only 0.66 mL of urine is required for a full scan. The repeatability of this UNE-PTR-MS was evaluated, and the relative standard deviations of 5 independent determinations were between 4.62% and 5.21%. Lastly, the UNE-PTR-MS was applied for detection of methanol, acetaldehyde, and acetone in real human urine to test matrix effects, yielding relative recoveries of between 88.39% and 94.54%. These results indicate the UNE-PTR-MS can be used for the rapid detection of VOCs in a drop of urine and has practical potential for diagnosing disease or toxin exposure.

Dermal and pulmonary absorption of ethanol from alcohol-based hand rub.

BACKGROUND: Ethanol intoxication of healthcare workers (HCWs) using alcohol-based hand rubs (ABHRs) in the workplace is a potentially serious issue. This study quantified the level of ethanol absorption among HCWs after hygienic hand disinfection. METHODS: Eighty-six HCWs from Nancy University Hospital were tested before and after a 4-h shift. Participants used ABHR containing 70% ethanol. Levels of ethanol, acetaldehyde and acetate in blood and urine were determined using gas chromatography. A breathalyzer was used to measure the level of ethanol in expired air. RESULTS: Ethanol [mean concentration 0.076 (standard deviation 0.05) mg/L] was detected in the expired air of 28 HCWs 1-2 min post exposure. Ethanol, acetaldehyde and acetate were undetectable in blood after a 4-h shift, and urine tests were negative in all participants. CONCLUSION: Ethanol exposure from ABHR, particularly inhalation of vapours, resulted in positive breathalyzer readings 1-2 min after exposure. Dermal absorption of ethanol was not detected. Pulmonary absorption was detected but was below toxic levels.

Environmental tobacco smoke exposure among casino dealers.

OBJECTIVE: This study quantified casino dealers' occupational exposure to environmental tobacco smoke (ETS). METHODS: We measured casino dealers' exposure to ETS components by analyzing full-shift air and preshift and postshift urine samples. RESULTS: Casino dealers were exposed to nicotine, 4-vinyl pyridine, benzene, toluene, naphthalene, formaldehyde, acetaldehyde, solanesol, and respirable suspended particulates. Levels of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in urine increased significantly during an 8-hour work shift both with and without adjustment for creatinine clearance. Creatinine-unadjusted cotinine significantly increased during the 8-hour shift, but creatinine-adjusted cotinine did not increase significantly. CONCLUSIONS: Casino dealers at the three casinos were exposed to airborne ETS components and absorbed an ETS-specific component into their bodies, as demonstrated by detectable levels of urinary NNAL. The casinos should ban smoking on their premises and offer employee smoking cessation programs.

Measurement of free and bound alcohol metabolites and methanol in human biological samples--free and bound alcohol metabolites and methanol in acute alcohol administration.

There are almost no studies on the in vivo distribution kinetics of free and bound ethanol, alcohol metabolites (acetaldehyde and acetate) or the related substance, methanol, during alcohol oxidation. Thus, an acute alcohol administration experiment (alcohol consumption experiment) was carried out using volunteers (five healthy adult males; 2 flushers, 3 non-flushers), and distribution kinetics were investigated in biological samples (blood and urine). The levels of alcohol metabolites and methanol were measured as free compounds in blood samples and bound and free compounds in urine samples. The results showed an increase over time of free alcohol metabolites in both the flusher and non-flusher groups, followed by a subsequent decrease. In addition, free methanol increased over time. Both bound alcohol metabolites and bound methanol were found to increase over time. Based on these findings, levels of free and bound alcohol metabolites and methanol in the biological samples were found to increase relative to levels before consumption in both the flusher and non-flusher groups. This is thought to be due to the binding of alcohol metabolites and methanol to biological components and increases during ethanol oxidation. It was concluded that this is the mechanism by which ethanol, alcohol metabolites and methanol accumulate in the body as a result of chronic alcohol consumption, suggesting that it may be possible to use these compounds as markers of consumption by measuring these compounds in biological samples taken from alcohol abusers or alcoholics.

Use of acetaldehyde and methanol as markers of alcohol abuse and their measurement.

UNLABELLED: It is believed that ethanol metabolites (ethanol, acetaldehyde and acetate) are produced when alcohol is consumed in accumulation with the binding of biological components. Additionally, it has been reported that ethanol metabolites and methanol present in the blood and urine are bound to biological components in habitual alcohol drinkers, even when alcohol has not been consumed. Consequently, with the purpose of investigating the potential for effectively using ethanol metabolite and methanol as markers of alcohol abuse, acetaldehyde level was measured in blood hemoglobin samples, and ethanol, acetaldehyde and methanol levels were measured in urine samples in healthy adult males (volunteers: control group) and alcoholics that had not consumed alcohol. Simultaneously, investigations were carried out on the genetic analysis of ADH and ALDH enzymes that participate in ethanol metabolism. Acetaldehyde levels were found to be significantly higher in alcoholics than in the volunteers for the period of 2 to 3 months after admission. In urine samples, acetaldehyde level (bound) was significantly higher in the period of 2 to 3 months after admission, and methanol level (free and bound) was significantly higher within a period of 1 month after admission. A correlation between alcoholics and genotype was found with regard to the distribution of ADH2 and ALDH2 genotypes ( VOLUNTEERS: ADH2 2-2 type, 81%; ALDH2 1-1 type, 61.9%; Alcoholics: ADH2 2-2 type, 39.6%; ALDH2 1-1 type, 84.9%). Based on these results, alcoholism or alcohol abuse might be predicted by concentration of acetaldehyde as well as methanol during abstinence. It also might be used as markers of alcohol abuse.

3-Penten-2-one, a novel aldehyde adduct, is a biomarker for increased acetaldehyde in urine.

Diagnostic profiling of urine for volatile compounds of around 400 patients using headspace solid-phase microextraction (HS-SPME) in alkaline conditions identified 3-penten-2-one (approximately 1 to >6.3 micromol/L) in 26 patients. Five were in barbiturate coma. 3-Penten-2-one, previously of unknown origin, was shown to be formed by aldol condensation of acetaldehyde with acetone or acetoacetate during analysis. Semi-quantification of acetaldehyde using in-fibre derivatisation HS-SPME, showed high concentrations in five urine (33-348 micromol/L) and two plasma (17 and 43 micromol/L) samples. Hence, urinary 3-penten-2-one is a useful biomarker for increased accumulation of acetaldehyde during abnormal metabolic stress.

ALDH2 and CYP2E1 genotypes, urinary acetaldehyde excretion and the health consequences in moderate alcohol consumers.

Deficiencies in mitochondrial low-Km aldehyde dehydrogenase (ALDH2) activity, and consequently high blood acetaldehyde levels, have been suggested to relate to various diseases in Japanese, including esophageal cancer. In the present study, 200 men aged 35-59 years randomly selected from an occupational population were analyzed for the association of ALDH2 genotypes and cytochrome P450-2E1 (CYP2E1) genotypes with the urinary excretion of acetaldehyde (which is bound to some chemicals in the urine) and with common alcohol-related health consequences. Urinary acetaldehyde excretion was increased, reflecting increased alcohol consumption even in this moderate alcohol-consuming population. Neither the ALDH2 nor the CYP2E1 genotypes showed significant influence on the elevation of urinary acetaldehyde excretion. Neither these genotypes nor urinary acetaldehyde concentration significantly affected blood pressure, serum aspartate aminotransferase and gamma-glutamyl transferase activities, or serum HDL-cholesterol and lipid peroxide concentrations. It was concluded that acetaldehyde accumulates in moderate alcohol consumers irrespective of ALDH2 and CYP2E1 genotype, and that the implications of these genotypes and acetaldehyde accumulation in terms of common alcohol-related health consequences were obscure. The results also suggest that the carcinogenicity of acetaldehyde on esophageal mucosa depends greatly upon repeated exposure to high blood acetaldehyde, even through transient rather than chronic exposure.

[Acetaldehyde and some biochemical parameters in alcoholic intoxications]. / Atsetal'degid i nekotorye biokhimicheskie parametry pri alkogol'nykh intoksikatsiiakh.

The need in comprehensive gas chromatography and biochemistry examinations is grounded for cadaver expertise in order to cope with issues related with alcoholic intoxication. Descriptions of 3 examination methods of biological fluids are elucidated, i.e. gas chromatography, electrophoresis and fixing of a degree of endogenous intoxication. The concentration of acetaldehyde in 3 body media (blood, urine and liquor) are analyzed in detail; the isoenzyme spectra of lactate-, alcohol- and aldehyde dehydrogenase as well as the contents of medium molecules in death of alcohol poisonings and due to mechanical trauma are also in the focus of attention.

[The diagnostic value of determining the level of acetaldehyde in blood, urine, and cerebrospinal fluid]. / Diagnosticheskoe znachenie opredeleniia atsetal'degida v krovi, moche i likvore.

The contents of acetaldehyde (AA) in biological fluids obtained from the dead with the confirmed lethality causes, i.e. ischemic heart disease (IHD), alcoholic cardiomyopathy (ACMP) and mechanical traumas (MT), were examined on an actual forensic-medical material (AFMM). 14 death cases of males, aged 18 to 45, were studied. The method of gas-liquid chromatography (GLC), or rather its variation of vaporphase analysis, was used to state the presence and to assess the concentration of acetaldehyde. The results revealed differences between concentrations of acetaldehyde in the examined groups depending on the presence or absence of alcoholemia. Thus, the AA concentrations were found in trace quantities in the MT group free of alcoholic intoxication; while, when it was present in this group, the concentrations went up several-fold. A higher AA content was typical of the ACMP group in all examined subjects both with and without alcoholic intoxication. The final study results are suggestive of that the AA determination in blood, urine and liquor by GLC could be used, within the forensic medical practice, in assessing a severity degree of alcoholic intoxication while establishing the lethal outcome cause due to chronic pathologies and MT.

Oxidative mechanisms in the toxicity of chromium and cadmium ions.

Chromium and cadmium are widely used industrial chemicals. The toxicities associated with both metal ions are well known. However, less information is available concerning the mechanisms of toxicity. The results of in vitro and in vivo studies demonstrate that both cations induce an oxidative stress that results in oxidative deterioration of biological macromolecules. However, different mechanisms are involved in the production of the oxidative stress by chromium and cadmium. Chromium undergoes redox cycling, while cadmium depletes glutathione and protein-bound sulfhydryl groups, resulting in enhanced production of reactive oxygen species such as superoxide ion, hydroxyl radicals, and hydrogen peroxide. These reactive oxygen species result in increased lipid peroxidation, enhanced excretion of urinary lipid metabolites, modulation of intracellular oxidized states, DNA damage, membrane damage, altered gene expression, and apoptosis. Enhanced production of nuclear factor-kappaB and activation of protein kinase C occur. Furthermore, the p53 tumor suppressor gene is involved in the cascade of events associated with the toxicities of these cations. In summary, the results clearly indicate that although different mechanisms lead to the production of reactive oxygen species by chromium and cadmium, similar subsequent mechanisms and types of oxidative tissue damage are involved in the overall toxicities.

Blood and urinary levels of ethanol, acetaldehyde, and C4 compounds such as diacetyl, acetoin, and 2,3-butanediol in normal male students after ethanol ingestion.

Aldehyde dehydrogenase (ALDH) isozyme 2 genes were determined in 15 students. Of these subjects, five healthy male students were administered 0.4 kg/kg ethanol. One subject was defective in aldehyde dehydrogenase 2 (ALDH2), two had normal ALDH2, and the other two were hetero type. After the intake of alcohol, the concentration of ethanol, acetaldehyde, and C4 compounds in blood and urine were determined. The student with the inactive form of ALDH2 was flushed and his levels of 2,3-butanediol and acetaldehyde in blood and urine were found to be the highest.

Deamination of methylamine and aminoacetone increases aldehydes and oxidative stress in rats.

Semicarbazide-sensitive amine oxidase (SSAO)-mediated deamination of methylamine and aminoacetone in vitro produces carbonyl compounds, such as formaldehyde and methylglyoxal, which have been proposed to be cytotoxic and may be responsible for some pathological conditions. An HPLC procedure was developed to assess different aldehydes, which were derivatized with 2,4-dinitrophenylhydrazine (DNPH). We have demonstrated in vivo deamination of methylamine and aminoacetone by examining the excretion of formaldehyde and methylglyoxal, respectively, in rats. Following chronic administration of methylamine, the urinary level of malondialdehyde (MDA), an end product of lipid peroxidation, was also found to be substantially increased. A selective SSAO inhibitor blocked the increase of MDA. The results support the idea that increased SSAO-mediated deamination of methylamine and aminoacetone can be a potential cytotoxic risk factor.

Determinations of free and bound ethanol, acetaldehyde, and acetate in human blood and urine by headspace gas chromatography.

The improved PCA method leads to accurate measurement of ethanol, acetaldehyde, and acetate in blood and urine by headspace gas chromatography. It is important to prevent the formation of artifactual acetaldehyde from coexistent ethanol. The column used for detection of alcohol metabolites was the fused silica glass capillary column bonded with PEG-20M or the fused silica glass capillary column of Pora PLOT Q. In bound alcohol metabolites, recent measurements of hemoglobin-associated acetaldehyde in blood, and ethanol conjugate and acetaldehyde conjugate in urine are reviewed and described as a marker of alcohol abuse.

Subchronic effects of smokeless tobacco extract (STE) on hepatic lipid peroxidation, DNA damage and excretion of urinary metabolites in rats.

The oral use of moist smokeless tobacco products (snuff) is causally associated with cancer of the mouth, lip, nasal cavities, esophagus and gut. The mechanism by which smokeless tobacco constituents produce genetic and tissue damage is not known. Recent studies in our laboratories have shown that an aqueous extract of smokeless tobacco (STE) activates macrophages with the resultant production of reactive oxygen species (ROS), including nitric oxide. Furthermore, the administration of acute doses of STE (125-500 mg/kg) to rats induces dose dependent increases in mitochondrial and microsomal lipid peroxidation, enhances DNA single strand breaks, and significantly increases the urinary excretion of the lipid metabolites malondialdehyde, formaldehyde, acetaldehyde and acetone. Since the use of tobacco is a chronic process, the effects of an aqueous extract of STE in rats following low dose exposure were examined. Female Sprague-Dawley rats were treated orally with 25 mg STE/kg every other day for 105 days. The effects of subchronic treatment of STE on hepatic microsomal and mitochondrial lipid peroxidation and the incidence of hepatic nuclear DNA damage were assessed. Lipid peroxidation increased 1.4- to 3.3-fold in hepatic mitochondria and microsome with STE treatment between 0 and 105 days with respect to control animals while hepatic DNA single strand breaks increased up to 3.4-fold. Maximum increases in lipid peroxidation and DNA single strand breaks occurred between 75 and 90 days of treatment. Urinary excretion of the four lipid metabolites malondialdehyde, formaldehyde, acetaldehyde and acetone was monitored by high pressure liquid chromatography (HPLC) with maximum increases being observed between 60 and 75 days of treatment. The results clearly indicate that low dose subchronic administration of STE induces an oxidative stress resulting in tissue damaging effects which may contribute to the toxicity and carcinogenicity of STE.

Excretion kinetics of ifosfamide side-chain metabolites in children on continuous and short-term infusion.

Ifosfamide (IFO) requires metabolic activation by hydroxylation of the ring system to exert cytotoxic activity. A second metabolic pathway produces the cytostatically inactive metabolites 2-dechloroethyl-ifosfamide (2-D-IFO) and 3-dechloroethyl-ifosfamide (3-D-IFO) under release of chloroacetaldehyde. This side-chain metabolism has been suggested to be involved in CNS- and renal toxicity. The total urinary excretion of ifosfamide and its metabolites was investigated during 23 cycles in 22 children at doses ranging from 400 mg/m2 to 3 g/m2. The kinetics of the excretion were compared following short-term and continuous ifosfamide infusion at a dosage of 3 g/m2. IFO and side-chain metabolites were analyzed by gas chromatography, the active metabolites by indirect determination of acrolein (ACR) and IFO mustard (IFO-M) with the NBP test. 59+/-15% of the applied dose could be recovered in the urine, 23+/-9% as unmetabolized IFO. The main metabolite was 3-D-IFO (14+/-4%) followed by isophosphoramide mustard (IFO-M) (13+/-4%) and 2-D-IFO (8+/-3%). Neither the total amount recovered nor the excretion kinetics of ifosfamide and side-chain metabolites showed obvious schedule dependency. The excretion kinetics of side-chain metabolites as well as unmetabolized IFO were nearly superimposable on short-term and continuous infusion. Even after 1-hour infusion there was a lag of 3 - 6 hours until dechloroethylation became relevant. Therefore, differences in toxicity and efficacy cannot be explained by an influence of the application time on the metabolic profile of ifosfamide.

Induction of oxidative stress by chronic administration of sodium dichromate [chromium VI] and cadmium chloride [cadmium II] to rats.

Recent studies have demonstrated that both chromium (VI) and cadmium (II) induce an oxidative stress, as determined by increased hepatic lipid peroxidation, hepatic glutathione depletion, hepatic nuclear DNA damage, and excretion of urinary lipid metabolites. However, whether chronic exposure to low levels of Cr(VI) and Cd(II) will produce an oxidative stress is not shown. The effects of oral, low (0.05 LD50) doses of sodium dichromate [Cr(VI); 2.5 mg/kg/d] and cadmium chloride [Cd(II); 4.4 mg/kg/d] in water on hepatic and brain mitochondrial and microsomal lipid peroxidation, excretion of urinary lipid metabolites including malondialdehyde, formaldehyde, acetaldehyde and acetone, and hepatic nuclear DNA-single strand breaks (SSB) were examined in female Sprague-Dawley rats over a period of 120 d. The animals were treated daily using an intragastric feeding needle. Maximum increases in hepatic and brain lipid peroxidation were observed between 60 and 75 d of treatment with both cations. Following Cr(VI) administration for 75 d, maximum increases in the urinary excretion of malondialdehyde, formaldehyde, acetaldehyde, and acetone were 2.1-, 1.8-, 2.1-, and 2.1-fold, respectively, while under the same conditions involving Cd(II) administration approximately 1.8-, 1.5-, 1.9-, and 1.5-fold increases were observed, respectively, as compared to control values. Following administration of Cr(VI) and Cd(II) for 75 d, approximately 2.4- and 3.8-fold increases in hepatic nuclear DNA-SSB were observed, respectively, while approximately 1.3- and 2.0-fold increases in brain nuclear DNA-SSB were observed, respectively. The results clearly indicate that low dose chronic administration of sodium dichromate and cadmium chloride induces an oxidative stress resulting in tissue damaging effects that may contribute to the toxicity and carcinogenicity of these two cations.

Oxidative stress induced by chronic administration of sodium dichromate [Cr(VI)] to rats.

Chromium occurs in the workplace primarily in the valence forms Cr(III) and Cr(VI). Recent studies have demonstrated that sodium dichromate [Cr(VI)] induces greater oxidative stress as compared with Cr(III), as indicated by the production of reactive oxygen species by peritoneal macrophages and hepatic mitochondria and microsomes, and enhanced excretion of urinary lipid metabolites and hepatic DNA-single strand breaks (SSB) following acute oral administration of Cr(III) and Cr(VI). We have therefore examined the chronic effects of sodium dichromate dihydrate [Cr(VI); 10 mg (33.56 mumol)/kg/day] on hepatic mitochondrial and microsomal lipid peroxidation, enhanced excretion of urinary lipid metabolites including malondialdehyde (MDA), formaldehyde (FA), acetaldehyde (ACT), acetone (ACON) and propionaldehyde (PROP), and hepatic DNA damage over a period of 90 days. The maximal increases in hepatic lipid peroxidation and DNA damage were observed at approximately 45 days of treatment. Maximum increases in the urinary excretion of MDA, FA, ACT, ACON and PROP were 3.2-, 2.6-, 4.1-, 3.3- and 2.1-fold, respectively, while a 5.2-fold increase in DNA-SSB was observed. The results clearly indicate that chronic sodium dichromate administration induces oxidative stress resulting in tissue damaging effects which may contribute to the toxicity and carcinogenicity of hexavalent chromium.

Chromium-induced excretion of urinary lipid metabolites, DNA damage, nitric oxide production, and generation of reactive oxygen species in Sprague-Dawley rats.

Chromium and its salts induce cytotoxicity and mutagenesis, and vitamin E has been reported to attenuate chromate-induced cytotoxicity. These observations suggest that chromium produces reactive oxygen species which may mediate many of the untoward effects of chromium. We have therefore examined and compared the effects of Cr(III) (chromium chloride hexahydrate) and Cr(VI) (sodium dichromate) following single oral doses (0.50 LD50) on the production of reactive oxygen species by peritoneal macrophages, and hepatic mitochondria and microsomes in rats. The effects of Cr(III) and Cr(VI) on hepatic mitochondrial and microsomal lipid peroxidation and enhanced excretion of urinary lipid metabolites as well as the incidence of hepatic nuclear DNA damage and nitric oxide (NO) production were also examined. Increases in lipid peroxidation of 1.8- and 2.2-fold occurred in hepatic mitochondria and microsomes, respectively, 48 hr after the oral administration of 25 mg Cr(VI)/kg, while increases of 1.2- and 1.4-fold, respectively, were observed after 895 mg Cr(III)/kg. The urinary excretion of malondialdehyde (MDA), formaldehyde (FA), acetaldehyde (ACT) and acetone (ACON) were determined at 0-96 hr after Cr administration. Between 48 and 72 hr post-treatment, maximal excretion of the four urinary lipid metabolites was observed with increases of 1.5- to 5.4-fold in Cr(VI) treated rats. Peritoneal macrophages from Cr(VI) treated animals 48 hr after treatment resulted in 1.4- and 3.6-fold increases in chemiluminescence and iodonitrotetrazolium reduction, indicating enhanced production of superoxide anion, while macrophages from Cr(III) treated animals showed negligible increases. Increases in DNA single strand breaks of 1.7-fold and 1.5-fold were observed following administration of Cr(VI) and Cr(III), respectively, at 48 hr post-treatment. Enhanced production of NO by peritoneal exudate cells (primarily macrophages) was monitored following Cr(VI) administration at both 24 and 48 hr post-treatment with enhanced production of NO being observed at both timepoints. The results indicate that both Cr(VI) and Cr(III) induce an oxidative stress at equitoxic doses, while Cr(VI) induces greater oxidative stress in rats as compared with Cr(III) treated animals.