Mutagenicity of malonaldehyde, a decomposition product of peroxidized polyunsaturated fatty acids.

Incubation of histidine requiring auxotrophs of the bacterium Salmonella typhimurium with malonaldehyde, a three-carbon dialdehyde, produced an increased number of revertants in specific strains. Mutagenesis was only observed in frameshift mutants with normal excision repair and did not occur in those base-pair substitution mutants tested. The results are consistent with the cross-linking of bacterial DNA by malonaldehyde leading to mutagenesis expressed through the error-prone repair system.

Ozone and vitamin E.

Vitamin E deficiency in rats is associated with a greater susceptibility to lethal levels of ozone. Exposure of rats to sublethal ozone concentrations produces an accelerated decline in serum vitamin E levels. These findings are consistent with the possibility that lipid peroxidation is a mechanism of ozone toxicity.

Dietary vitamin D3 restriction influences tumor growth, but not the ability to generate an antigen-specific immune response in OTII transgenic mice.

Initially, we wanted to know whether dietary vitamin D(3) restriction would influence growth and metastasis of the 4T1 murine mammary carcinoma. We confirmed serum 25(OH)D levels were modulated by dietary vitamin D(3) restriction, mice were healthy, and when challenged with the 4T1 tumor alterations in tumor growth, but not metastasis were evident. Tumors grew more rapidly in mice on the vitamin D(3) restricted diet. To delineate whether dietary vitamin D(3) restriction influenced the ability to generate an antigen-specific immune response we used OTII transgenic mice which express a T cell receptor specific for ovalbumin. We found that dietary vitamin D(3) restriction did not influence the health of OTII mice, the number of circulating CD3/CD4(+), CD3/CD8(+), CD4/CD25(+) T cells, nor the ability to generate CD11c(+) bone-marrow derived dendritic cells. T cells from OTII mice maintained on the vitamin D(3) restricted diet also exhibited no significant alterations in proliferative capacity or ability to secrete IFN-gamma or IL-4 in an antigen-specific manner. Yet, EL-4 tumors grew more rapidly in OTII mice on the vitamin D(3) restricted diet. These data show that dietary vitamin D(3) restriction impacts tumor growth, but not the ability to generate an antigen-specific immune response.

Early effects of low benzene exposure on blood cell counts in Bulgarian petrochemical workers.

OBJECTIVES: Only few studies have examined early hematological effects in human populations exposed to low benzene levels and their findings are controversial. We evaluated hematological outcomes (WBC, neutrophils, lymphocytes, monocytes, eosinophils, basophils, RBC, Hb, HCT MCV, platelets and MPV) in a population of 153 Bulgarian petrochemical workers exposed to benzene (range 0.01-23.9 ppm) and 50 unexposed subjects. METHODS: Written informed consent was obtained and a self-administered questionnaire used to collect information on current smoking habits, lifestyle, and occupational activities. Exposure assessment was based on personal monitoring sampling the day before phlebotomy. Urinary trans-trans-muconic acid (t,t-MA) was determined at the beginning and end of the work shift. Based on individual airborne benzene measurements, study subjects were categorized in three exposure categories (referents, <1 and > or =1 ppm). Mean values of each hematologic outcomes in each exposure category were compared with the referent group using a multiple linear regression model adjusted for age, gender, current smoking habits and environmental toluene level. The influence of the CYP2E1 (RsaI and DraI) and NQO1 609C>T genetic polymorphisms on differential hematological parameters was also investigated. RESULTS: No dose-response effect was observed for most of the examined hematological outcomes (WBC, lymphocytes, neutrophils, monocytes, RBC, Hb, HCT, MCV, platelets and MPV). The eosinophil count was inversely related to benzene exposure only among smokers. Conversely, basophils increased with increasing exposure. No effect on benzene hematotoxicity was found for any of the investigated polymorphisms. CONCLUSION: In our study we did not find a decline in WBC and lymphocytes related to benzene exposure. A myeloproliferative effect of benzene is highly unlikely to explain the observed reduction in eosinophils and increase in basophils as it would lead to a concordant depression in all granulocyte subpopulations. Whether benzene effects at low doses are present in Caucasian populations remains uncertain, thus warranting further investigations.

Erythropoietic protoporphyria: lipid peroxidation and red cell membrane damage associated with photohemolysis.

The mechanism by which long wavelength ultraviolet light hemolyzes red cells obtained from patients with erythropoietic protoporphyria (EPP) was investigated. Previous studies had suggested that irradiation of these red cells with wavelengths of light capable of eliciting dermatological manifestations led to oxygen-dependent colloid osmotic hemolysis through the formation of peroxides. In the present report, lipid peroxidation during in vitro irradiation of EPP red cells with long ultraviolet light was demonstrated by: (a) the formation of 2-thiobarbituric acid reactants; (b) the presence of conjugated diene bonds in red cell lipid; and (c) the selective loss of unsaturated fatty acids proportional to the number of carbon-carbon double bonds in each. Irradiation of EPP red cells was also shown to result in the formation of hydrogen peroxide.Before photohemolysis there was a decline in cell membrane sulfhydryl groups and a loss in activity of the cell membrane enzyme acetylcholinesterase. These parameters provide further evidence suggesting that the cell membrane is a primary site of the photohemolytic effect of long ultraviolet light in EPP red cells. Further evaluation of the radiation-induced inactivation of EPP red cell acetylcholinesterase was performed by radiating mixtures containing bovine erythrocyte acetylcholinesterase and protoporphyrin IX. These studies revealed that the rate of decline in enzyme activity is accelerated by the addition of linoleic acid, an unsaturated fatty acid, but not by palmitic acid, a saturated fatty acid. Partial protection against both photohemolysis and acetylcholinesterase decline is provided by alpha-to-copherol. This lipid antioxidant loses its activity during the irradiation of EPP red cells suggesting that it is utilized in this process.

Spectrofluorescent detection of in vivo red cell lipid peroxidation in patients treated with diaminodiphenylsulfone.

In the absence of vitamin E deficiency, red cell lipid peroxidation has not been clearly demonstrated in freshly drawn blood obtained from patients with various hemolytic anemias despite indirect evidence that oxidative decomposition of cell membrane unsaturated fatty acids occurs in these particular hemolytic states. Recent studies have indicated that malonaldehyde, a decomposition product of oxidized polyunsaturated fatty acids, is able to covalently cross-link the amino groups of protein or lipid resulting in a fluorescent compound. In the present study we have utilized spectrofluorescent technique to assess whether such fluorescence is present in red cell lipid extracts in association with lipid peroxidation. In vitro red cell lipid peroxidation produced by ultraviolet radiation or the oxidant gas ozone was associated with the development of a fluorescent peak (excitation maximum 360 nm; emission maximum 440 nm) in lipid-containing red cell extracts Similar fluorescence was observed after incubation of red cells with malonaldehyde or with malonaldehyde-containing extracts of peroxidized red cell lipid. Spectrofluorescent evaluation of chloroform: isopropanol extracts obtained from the freshly drawn red cells of six patients receiving the oxidant hemolytic drug diaminodiphenylsulfone also revealed a peak at 440 nm which ranged from 39 to 78 U. In contrast, the levels in samples obtained from 11 hematologically normal subjects were 17-27 fluorescence U. No evidence for an increase in blood levels of free malomaldehyde was observed using the 2-thiobarbituric acid test which is the most commonly performed assay of lipid peroxidation. Serum vitamin E levels were within the normal range. Density separation indicated that the bulk of the fluorescence was present in older red cells. A similar fluorescent peak was also observed in lipid-containing extracts of red cells obtained from rabbits repeatedly injected with phenylhydrazine. The finding of fluorescent spectra consistent with the cross-linking of aminolid by malonaldehyde in the red cells of patients receiving diaminodiphenylsulfone indicates that in vivo red cell lipid peroxidation does occur in the absence of vitamin E deficiency.

Studies on pathways of ring opening of benzene in a Fenton system.

Ring-opened products of benzene metabolism have been postulated to play a role in hematotoxicity and leukemogenesis. The reaction of benzene in the Fenton system was reexamined to determine the presence of compounds which might serve as intermediates in the formation of trans, trans-muconaldehyde (MUC), a microsomal hematotoxic metabolite of benzene. Benzene dihydrodiol (DHD) was found in this system based on coelution with authentic standard, ultraviolet (UV) absorption characteristics, and molecular weight. Incubation of DHD in the Fenton system resulted in the formation of phenol (PH), catechol (CAT), and products which reacted with thiobarbituric acid to form chromogens absorbing at 495 nm and 532 nm, consistent with products containing an alpha, beta-unsaturated aldehyde group. However, muconaldehyde was not detected in the Fenton system incubated with DHD, indicating that MUC is not formed via ring opening of DHD. When benzene was incubated in the Fenton system, MUC, cis,trans-muconaldehyde, PH, hydroquinone (HQ), and CAT were identified. Identification of cis,trans-muconaldehyde, an isomer which can quickly rearrange to MUC, suggests that cis,cis-muconaldehyde is originally formed from benzene and converted to cis,trans- and then trans,trans-muconaldehyde.

Degradation of soluble collagen by ozone or hydroxyl radicals.

Collagen exposed to ozone or hydroxyl radicals was degraded in a time- and dose-dependent manner. This degradation was inhibited by free radical scavengers. Furthermore, lower levels of these oxidants did not degrade the molecule, but caused it to become susceptible to proteolytic degradation. We suggest an alternative mechanism by which oxygen-derived free radicals participate in the destruction of extracellular matrix observed during acute lung injury by oxidant gas, in addition to the commonly accepted proteinase-antiproteinase theory of lung injury.

Electrophysiological evidence for nicotinic and muscarinic modulation of spinal cord reflexes.

Intravenous administration of physostigmine has been found to produce dose- and time-dependent alterations of the lumbar monosynaptic and polysynaptic reflexes in spinal cats. A small dose of physostigmine (0.8 mg/kg) enhanced the monosynaptic reflex for 3 hr, while a larger dose (2.0 mg/kg) produced a pattern of initial depression which peaked at 5 min, followed by an increase which was maintained for 3 hr after injection. Both doses of physostigmine were found to produce a similar pattern of enhancement of the polysynaptic reflex. Atropine and mecamylamine antagonized the increase of the monosynaptic reflex produced by both doses of physostigmine. The initial depression of the monosynaptic reflex produced by the large dose of physostigmine was blocked by mecamylamine, but unaffected by atropine. The enhancement of the polysynaptic reflex, produced by both doses of physostigmine, was antagonized by atropine but not by mecamylamine. In addition, nicotine and oxotremorine were found to mimic the initial effects of physostigmine in the presence of the appropriate antagonist. These data show that both muscarinic and nicotinic receptors are involved differentially in the modulation of spinal reflexes and that physostigmine had an additional late effect which consistently occurred at 20 min after administration.

The role of GABA and glycine in the physostigmine-induced alterations of spinal cord reflexes.

The purpose of this study was to determine which inhibitory pathway(s) mediate the alterations in the monosynaptic (MSR) and polysynaptic (PSR) reflexes after two different doses of physostigmine. It was found previously that 0.8 mg/kg physostigmine facilitated the MSR and 2.0 mg/kg initially depressed and then facilitated the MSR. Both doses facilitated the PSR. In this study, the animals were pretreated with either strychnine (0.1 mg/kg) or bicuculline (0.5 mg/kg), prior to the administration of either dose of physostigmine. It was found that both strychnine and bicuculline blocked the facilitation produced by the small dose of physostigmine, while bicuculline alone blocked the depression of the MSR produced by the large dose of physostigmine. Strychnine partially blocked the effects of both doses of physostigmine on the PSR, while bicuculline only partially blocked the effects of the small dose of physostigmine. These data suggest that the depression of the MSR was the result of a GABA-mediated pathway, while the facilitation of MSR involved both glycine and GABA.

Stimulation of human polymorphonuclear leukocyte superoxide anion radical production by tumor promoters.

Comparison was made of the ability of the potent tumor promoter phorbol myristate acetate (PMA), as well as less active PMA analogs and non-phorbol ester tumor promoters, to stimulate superoxide anion radical (O-.2) production by human polymorphonuclear leukocytes (PMN). The rate of O-.2 production was found to correlate with the tumor-promoting activity of the phorbol esters as opposed to their inflammatory activity. Mezerein and telocidin B were slightly better stimulators of O-.2 production than PMA. Acetic acid was inactive. These data are discussed in terms of a possible role for O-.2 and other reactive oxygen species in tumor promotion.

Iron-stimulated ring-opening of benzene in a mouse liver microsomal system. Mechanistic studies and formation of a new metabolite.

In the present study, we investigated the mechanism(s) of ring-opening of benzene in a mouse liver microsomal system in the presence of Fe2+.HPLC analysis based on coelution with authentic standards and on-line UV spectra obtained using a diode array detector indicated that benzene is metabolized to phenol, hydroquinone (HQ), trans,trans-muconaldehyde (muconaldehyde, MUC), 6-oxo-trans,trans-2,4-hexadienoic (COOH-M-CHO), 6-hydroxy-trans,trans-2,4-hexadienal (CHO-M-OH), and 6-hydroxy-trans,trans-2,4-hexadienoic acid (COOH-M-OH). CHO-M-OH was confirmed by mass spectrometry. Muconaldehyde was also metabolized to CHO-M-OH, COOH-M-CHO and COOH-M-OH, in the same microsomal system. The inhibition of muconaldehyde metabolism by microsomes in the presence of pyrazole indicates that there is cytosolic alcohol dehydrogenase (ADH) activity in the microsomes. Metabolism by contaminating ADH of muconaldehyde formed during microsomal incubation of benzene could be involved in the formation of CHO-M-OH and COOH-M-OH. The ring-opening of benzene was stimulated by added Fe2+. Hydrogen peroxide was produced in the microsomal system and consumed in the presence of added Fe2+. Addition of catalase inhibited the formation of ring-opened products, while superoxide dismutase increased their formation in the presence of azide. Singlet oxygen scavengers, i.e. histidine, deoxyguanosine, Tris and azide (at concentrations above 1.0 mM), dramatically decreased the ring-opening of benzene. Hydroxyl radical scavengers, DMSO, mannitol and formate, but not ethanol, also decreased the ring-opening of benzene. The data indicate that Fenton chemistry plays an important role in benzene ring-opening by microsomes. An unknown peak with UV absorption maxima at 275 and 345 nm was also detected. Based on pH sensitivity of the UV spectrum, the reactivity with thiobarbituric acid (giving a chromogen with absorption maximum at 532 nm) and the molecular weight (126), this compound was identified tentatively as alpha- or beta-hydroxymuconaldehyde.

Comparative metabolism of benzene and trans,trans-muconaldehyde to trans,trans-muconic acid in DBA/2N and C57BL/6 mice.

Our laboratory recently identified trans,trans-muconaldehyde (MUC), a six-carbon diene dialdehyde, as a hematotoxic microsomal metabolite of benzene (Latriano et al., Proc Natl Acad Sci USA 83: 8356-8360, 1986). We also showed that MUC is metabolized in vitro to trans,trans-muconic acid (MA), a six-carbon diene dicarboxylic acid and known urinary metabolite of benzene. To elucidate further the role of ring-opened metabolites in benzene toxicity, the metabolism of benzene and MUC was examined in the benzene sensitive DBA/2N mouse strain and the less benzene sensitive C57BL/6 strain. A sensitive assay for urinary MA analysis was developed. The percent of benzene dose excreted as urinary MA within the first 24 hr after treatment decreased with an increase in benzene dose, i.e. from 9.8 to 0.4% in DBA/2N mice and from 17.6 to 0.2% in C57BL/6 mice treated with 0.5 to 880 mg/kg benzene. DBA/2N mice excreted significantly (P less than or equal to 0.05) more MA compared with C57BL/6 mice after treatment with hematotoxic benzene doses (220-880 mg/kg). At low benzene doses (0.5 to 2.5 mg/kg), C57BL/6 mice excreted significantly (P less than or equal to 0.05) more MA compared with DBA/2N mice. There were no significant differences in the metabolism of MUC to MA between the two strains after treatment with 0.5 to 3.0 mg/kg. Furthermore, mice from both strains excreted similar amounts of muconic acid when treated with 0.7 to 7.1 mg/kg MA. These results are consistent with the hypothesis that reactive ring-opened metabolites such as trans,trans-muconaldehyde play a role in benzene hematotoxicity. Sensitivity towards benzene may be due, in part, to increased metabolism to ring-opened compounds.

Inhibition by reactive aldehydes of superoxide anion radical production from stimulated polymorphonuclear leukocytes and pulmonary alveolar macrophages. Effects on cellular sulfhydryl groups and NADPH oxidase activity.

Alpha,beta-unsaturated aldehydes such as acrolein (ACR) and crotonaldehyde (CRO) have been shown previously in our laboratory to inhibit the production of superoxide anion radical (O2-) by stimulated phagocytic cells in vitro in a dose-related manner. Based on the known reactivity of these compounds towards cellular sulfhydryls (SH), the present studies were aimed at investigating cellular SH status in relation to O2- production. Plasma membrane surface SH groups were measured using carboxypyridinedisulfide and monitoring the resultant formation of mixed disulfides through assay of thione released into the supernatant fraction. Intracellular non-protein sulfhydryls were measured using 5,5'-dithiobis-2-nitrobenzoic acid. In both human polymorphonuclear leukocytes (PMN) and rat pulmonary alveolar macrophages (PAM) there was a dose-related decrease in surface SH and soluble SH after ACR and CRO treatment. Propionaldehyde, a three-carbon saturated aldehyde, was without effect. The decrease in surface SH was greater than the decrease in soluble SH. In addition, in PMN and PAM preincubated with 5-40 microM ACR, there was a dose-related inhibition in the rate of O2- production with no effect on the lag time as measured by cytochrome c reduction. In stimulated PMN, there was a dose-related decrease in the rate after addition of 5-40 microM ACR. These data suggest that changes in SH status by reactive aldehydes can modulate the activity of the plasma membrane NADPH oxidase responsible for O2- production.

Health effects of the gas-aerosol complex. Report to Special Committee on Health and Ecological Effects of Increased Coal Utilization.

Combustion products derived from the burning of coal are definitely capable of producing adverse human health effects. No single component of the combustion product mixture is solely responsible. Rather, effects are due to a group of compounds, both gases and aerosols, in the effluents of stationary source combustion processes. Although incompletely defined, the individual components of the gas-aerosol complex appear to be capable of interacting both in terms of atmospheric chemistry and health effects. The three primary air quality standards pertinent to regulating coal combustion all represent to some extent indirect, although reasonable, measures of this gas-aerosol complex. As a group, these standards appear to be adequate to protect human health. Conventional toxicological considerations suggest that the adverse health effects of any necessary increase in coal combustion effluents would be greatest per unit of coal in those areas which are most heavily populated and have the highest preexisting levels of the gas-aerosol complex. In order to decrease the degree of uncertainty for future decisions of this type, it is important that prospective epidemiological and air monitoring studies be initiated in conjunction with any large scale introduction of coal use.

Combined exposure to ozone and nitrogen dioxide.

A study of rats acutely exposed to ozone (0.5--2.0 ppm) or nitrogen dioxide (2--20 ppm) for 2 hr and sacrificed immediately thereafter shows little similarity in the individual biochemical effects of these pollutants. No evidence of nitrogen dioxide-induced lipid peroxidation was observed. Of interest is the finding that inhalation of nitrogen dioxide increases the extent to which concanavalin A agglutinates alveolar macrophages while ozone has exactly the opposite effect.

Combined exposure to ozone and nitrogen dioxide.

The combined effects of ozone and nitrogen dioxide were assessed in an in vitro test system utilizing human red cells. In general, these two pollutants had additive effects on the parameters measured which included osmotic fragility, acetylcholinesterase activity, lipid peroxidation, reduced glutathione and methemoglobin levels. However, at lower pollutant doses a synergistic increase in lipid peroxides was noted while at higher doses the effect became less than additive. Further studies of this observation suggested that ferrous hemoglobin potentiates ozone-induced lipid peroxidation while methemoglobin, resulting primarily from nitrogen dioxide, inhibits this process. Ozone was also found to potentiate the methemoglobinemic effects of nitrogen dioxide, particularly in sequential studies in which ozone exposure preceded nitrogen dioxide. Inasmuch as the effects of these two pollutants vary from protective to synergistic depending on the pollutant concentration, duration and sequence of exposure, as well as on the parameter assayed, it would appear that the approach to in vivo study of the combined effects of ozone and nitrogen dioxide should be aimed at simulating ambient conditions as closely as possible.

The precautionary principle and scientific research are not antithetical.

The Precautionary Principle is intended to protect human health and the environment. To serve these goals effectively, precautionary action must be coupled with concurrent research to decide whether the action taken is in fact protective.

A public health context for residual risk assessment and risk management under the clean air act.

The 1990 amendments to the Clean Air Act required the EPA to institute new pollution control technology requirements for industrial sources of air pollution. In part because agreement could not be reached on the best way for the EPA to determine whether any significant risks to human health will remain after the technology controls are in place, the amendments also created a Commission on Risk Assessment and Risk Management and gave the commission a broad mandate to review and make recommendations concerning risk assessment and risk management in federal regulatory programs. In its March 1997 final report to Congress and the administration, the commission recommended a tiered approach to assessing such residual risks. That approach included the idea that when decisions about managing residual risks are made, emissions should be evaluated in the context of other sources of air pollution. Evaluating risks in their larger contexts is consistent with what the commission called a public health approach to environmental risk management. This paper describes the public health approach and how it applies to evaluating residual risks under the Clean Air Act.

The toxicology of benzene.

Benzene is metabolized, primarily in the liver, to a series of phenolic and ring-opened products and their conjugates. The mechanism of benzene-induced aplastic anemia appears to involve the concerted action of several metabolites acting together on early stem and progenitor cells, as well as on early blast cells, such as pronormoblasts and normoblasts to inhibit maturation and amplification. Benzene metabolites also inhibit the function of microenvironmental stromal cells necessary to support the growth of differentiating and maturing marrow cells. The mechanism of benzene-induced leukemogenesis is less well understood. Benzene and its metabolites do not function well as mutagens but are highly clastogenic, producing chromosome aberrations, sister chromatid exchange, and micronuclei. Benzene has been shown to be a multi-organ carcinogen in animals. Epidemiological studies demonstrate that benzene is a human leukemogen. There is need to better define the lower end of the dose-response curve for benzene as a human leukemogen. The application of emerging methods in biologically based risk assessment employing pharmacokinetic and mechanistic data may help to clarify the uncertainties in low-dose risk assessment.