Toxicology of simple and complex mixtures.

Humans are exposed to mixtures of chemicals, rather than to individual chemicals. From a public health point of view, it is most relevant to answer the question of whether or not the components in a mixture interact in a way that results in an increase in their overall effect compared with the sum of the effects of the individual components. In this article, options for the hazard identification and risk assessment of simple and complex chemical mixtures will be discussed. In addition, key research needed to continue the development of hazard characterization of chemical mixtures will be described. Clearly, more collaboration among toxicologists, model developers and pharmacologists will be necessary.

Intercellular communication and cell proliferation in precision-cut rat liver slices: effect of medium composition and DDT.

Gap junctional intercellular communication (GJIC) and cell proliferation were studied in control and 1,1'-bis(p-chlorophenyl)-2, 2,2,-trichloroethane (DDT) treated precision-cut liver slices of rat by evaluating connexin 32 (Cx32) expression and 5-bromo-2'-deoxyuridine (BrdU) incorporation. In addition, the effect of different culture media (RPMI and WME) on control and DDT influenced Cx32 expression and cell proliferation was determined. Cx32 expression in control precision-cut liver slices was maintained during 8 h of culturing, but decreased after prolonged culturing. Control cell proliferation was significantly higher when WME was used as culture medium than when RPMI was used. In slices treated with DDT Cx32 expression was decreased. In slices cultured in RPMI medium, this decrease preceded a dose-dependent increase in cell proliferation. These results show the usefulness of precision-cut liver slices in studying cellular proliferation and intercellular communication.

Toxicology of chemical mixtures: international perspective.

This paper reviews major activities outside the United States on human health issues related to chemical mixtures. In Europe an international study group on combination effects has been formed and has started by defining synergism and antagonism. Successful research programs in Europe include the development and application of statistically designed experiments combined with multivariate data analysis and modeling in vitro and in vivo studies on a wide variety of chemicals such as petroleum hydrocarbons, aldehydes, food contaminants, industrial solvents, and mycotoxins. Other major activities focus on the development of safety evaluation strategies for mixtures such as the use of toxic equivalence factors or alternatives such as the question-and-answer approach, fractionation followed by recombination of the mixture in combination with a mixture design, and quantitative structure-activity relationship analysis combined with lumping analysis and physiologically based pharmacokinetic/pharmacodynamic modeling for studying complex mixtures. A scheme for hazard identification and risk assessment of complex mixtures and a consistent way to generate total volatile organic compound values for indoor air have also been developed. Examples of other activities are carcinogenicity studies on complex mixtures (petroleum middle distillates, foundry fumes, pesticides, heterocyclic amines, diesel exhaust, solid particles), neurotoxicity studies of mixtures of solvents alone or in combination with exposure to physical factors, and toxicity studies of outdoor air pollutants, focusing on particulates. Outside the United States, toxicologists and regulators clearly have a growing interest in the toxicology and risk assessment of chemical mixtures.

Statistically designed experiments to screen chemical mixtures for possible interactions.

For the accurate analysis of possible interactive effects of chemicals in a defined mixture, statistical designs are necessary to develop clear and manageable experiments. For instance, factorial designs have been successfully used to detect two-factor interactions. Particularly useful for this purpose are fractionated factorial designs, requiring only a fraction of all possible combinations of a full factorial design. Once the potential interaction has been detected with a fractionated design, a more accurate analysis can be performed for the particular binary mixtures to ensure and characterize these interactions. In this paper this approach is illustrated using an in vitro cytotoxicity assay to detect the presence of mixtures of Fusarium mycotoxins in contaminated food samples. We have investigated interactions between five mycotoxin species (Trichothecenes, Fumonisins, and Zearalenone) using the DNA synthesis inhibition assay in L929 fibroblasts. First, a central composite design was applied to identify possible interactive effects between mycotoxins in the mixtures (27 combinations from 5(5) possible combinations). Then two-factor interactions of particular interest were further analyzed by the use of a full factorial design (5 x 5 design) to characterize the nature of those interactions more precisely. Results show that combined exposure to several classes of mycotoxins generally results in effect addition with a few minor exceptions indicating synergistic interactions. In general, the nature of the interactions characterized in the full factorial design was similar to the nature of those observed in the central composite design. However, the magnitude of interaction was relatively small in the full factorial design.

Availability of polychlorinated biphenyls (PCBs) and lindane for uptake by intestinal Caco-2 cells.

Children may ingest contaminated soil from hand to mouth. To assess this exposure route, we need to know the oral bioavailability of the contaminants. Two determining steps in bioavailability of soil-borne contaminants are mobilization from soil during digestion, which is followed by intestinal absorption. The first step has been investigated in previous studies that showed that a substantial fraction of PCBs and lindane is mobilized from soil during artificial digestion. Furthermore, almost all contaminants are sorbed to constituents of artificial human small intestinal fluid (i.e., chyme), whereas only a small fraction is freely dissolved. In this study, we examine the second step using intestinal epithelial Caco-2 cells. The composition of the apical exposure medium was varied by addition of artificial chyme, bile, or oleic acid at similar or increasing total contaminant concentrations. The uptake curves were described by rate constants. The uptake flux seemed to be dose-dependent. Furthermore, different exposure media with similar total contaminant concentrations resulted in various uptake rates. This can be attributed to different freely dissolved concentrations and carrier effects. In addition, the large fractions of contaminants in the cells indicate that PCBs and lindane sorbed to bile, oleic acid, and digestive proteins contributed to the uptake flux toward the cells. These results can be extrapolated qualitatively to in vivo conditions. Because the sorbed contaminants should be considered available for absorption, the first step of mobilization from soil is the most important step for oral bioavailability of the presently investigated soil-borne contaminants.

Dietary iron lowers the intestinal uptake of cadmium-metallothionein in rats.

It has been shown that addition of extra calcium/phosphorus (Ca/P), zinc (Zn) and iron (Fe2+) to the diet results in a significant protection against cadmium (Cd) accumulation and toxicity in rats fed inorganic Cd salt. However, it is not clear whether the presence of these mineral supplements in the diet also protects against the Cd uptake from cadmium-metallothionein. The present study examines the influence of Ca/P, Zn and Fe2+ on the Cd disposition in rats fed diets containing either 1.5 and 8 mg Cd/kg diet as cadmium-metallothionein (CdMt) or as cadmium chloride (CdCl2) for 4 weeks. The feeding of Cd resulted in a dose-dependent increase of Cd in intestine, liver and kidneys. The total Cd uptake in liver and kidneys after exposure to CdMt was lower than after exposure to CdCl2. At the low dietary Cd level and after addition of the mineral supplement, the kidney/liver concentration ratio increased. However, this ratio was always higher with CdMt than with CdCl2, suggesting a selective renal disposition of dietary CdMt. The uptake of Cd from CdCl2 as well as from CdMt was significantly decreased by the presence of a combined mineral supplement of Ca/P, Zn and Fe2+. The protection which could be achieved was 72 and 75% for CdMt and 85 and 92% for CdCl2 after doses of 1.5 mg/kg and 8 mg/kg respectively. In a following experiment it was shown that the protective effect of the mineral mixture against CdMt was mainly due to the presence of Fe2+. It seems clear that Cd speciation and the mineral status of the diet have a considerable impact on the extent of Cd uptake in rats.

Toxicology of chemical mixtures: challenges for today and the future.

A major challenge for the toxicologist involved in safety evaluation of chemical mixtures is to test the hypothesis that as a rule exposure to mixtures of chemicals at (low) non-toxic doses of the individual chemicals is of no health concern. A series of repeated dose studies in rats with defined mixtures of chemicals with the same or different target organs revealed that exposure to a combination of chemicals compared with exposure to the individual compounds did not constitute an evidently increased hazard, provided each chemical was administered at a level similar to, or slightly lower than, its own 'No-Observed-Adverse-Effect-Level'. The results of subacute oral toxicity studies in rats with defined mixtures of nephrotoxicants with similar mode of action underlined the applicability of the additivity assumption for a mixture of chemicals with simple similar action. Safety evaluation of complex chemical mixtures is a challenge that can be tackled as follows: first, identify the (e.g. ten) most risky chemicals in the mixture, and, second, assess the hazard and the potential health risk of the mixture of the most risky chemicals, using procedures developed for defined mixtures. To identify interactions between individual compounds, a most promising testing strategy appeared to be a statistical approach using a fractional two-level factorial design. A challenge for today and the future is to gradually substitute mixture-oriented (real life-oriented) standard setting for (unrealistic) single chemical-oriented standard setting.

Identification of a reactive intermediate of furazolidone formed by swine liver microsomes.

Furazolidone (N-(5-nitro-2-furfurylidene)-3-amino-2-oxazolidone) is metabolized by swine liver microsomes under aerobic and anaerobic conditions (rate: 2.55 and 3.25 nmol/mg protein/min, respectively). Covalent binding to microsomal protein amounted aerobically to 0.29 nmol/mg protein/min. Of all amino acids tested, only addition of cysteine to the incubation mixture decreased microsomal protein binding of furazolidone, indicating that covalent binding may occur at protein thiol groups. Two known metabolites of furazolidone, 3-(4-cyano-2-oxobutylidene-amino)-2-oxazolidone and 2,3 dihydro-3-cyano-methyl-2-hydroxyl-5-nitro-1 alpha,2-di(2-oxo-oxazolidin-3-yl) iminomethyl-furo[2,3-b] furan, were minor metabolites. At least 50% of total metabolites is formed by swine liver microsomes via a reductive process of furazolidone as indicated by the formation of a furazolidone-mercaptoethanol conjugate after the addition of mercaptoethanol to the incubation mixture. The conjugate was identified as 3-(4-cyano-3-beta-hydroxyethylmercapto-2-oxobutylidene amino)-2-oxazolidone, indicating that the open-chain acrylonitrile-derivative is the reactive intermediate of furazolidone which also may be responsible for interaction with protein.

Toxicity studies in rats of simple mixtures of chemicals with the same or different target organs.

Mixtures of chemicals with different target organs or the same target organ but different target sites or different modes of action did not appear to be distinctly more hazardous than the individual chemicals, provided the dose level of each chemical in the mixture did not exceed its own 'No-Observed-Adverse-Effect Level'. Clearly, for such mixtures and exposure conditions the additivity assumption did not hold. However, the additivity rule appeared to be applicable to mixtures of chemicals with the same target organ and the same mechanism of action or receptor. Fractional 2-factorial study designs were found to be promising tools for examining possible combined actions or interactions of chemicals in a mixture.

Comparative toxicity and accumulation of cadmium chloride and cadmium-metallothionein in primary cells and cell lines of rat intestine, liver and kidney.

The protective role of metallothionein (Mt) in the toxicity of cadmium (Cd) is controversial, since Cd bound to Mt is more nephrotoxic than ionic Cd after parenteral exposure and less hepatotoxic than ionic Cd after oral exposure. This study compared the uptake and toxicity in vitro of CdCl(2) and two isoforms of rat cadmium-metallothionein (CdMt-1 and CdMt-2) using primary rat kidney cortex cells, primary rat hepatocytes, liver hepatoma cell line H-35, kidney epithelial cell line NRK52-E and intestinal epithelial cell line IEC-18. The molar ratio of Cd was 2.1 and 1.4 mol Cd/mol Mt for CdMt-1 and CdMt-2, respectively. Monolayer cultures were incubated for 22 hr with CdCl(2), CdMt-1 or CdMt-2 and Cd accumulation was examined at Cd levels of 0.25-10 muM-Cd. Cells exposed to CdCl(2) accumulated more Cd in 22 hr than cells exposed to an equimolar amount of CdMt. For CdCl(2) the Cd accumulation is directly related to the Cd concentration in the medium; however, for CdMt an increase in Cd concentration in the medium above 2 muM had no effect on the Cd accumulation in the cells. At Cd concentrations above 2 muM, therefore, the difference in Cd accumulation between CdCl(2) and CdMt was greater (5-6 times) than at concentrations below 2 muM (1-2 times). Cytotoxicity was examined in the Cd-concentration range from 0.25 to 100 muM by determining the lactate dehydrogenase (LDH) release in the medium and the neutral red uptake in the cells. Under these culture conditions CdCl(2) was at least 100 times more toxic than CdMt-1 or CdMt-2 in all cell types tested. Primary hepatocyte cultures were 10 times more sensitive (50% LDH release at 1-2 muM) to CdCl(2) intoxication than primary cultures of renal cortical cells or the intestinal cell line (50% LDH release at 10-20 muM). Hepatic and renal cell lines were less sensitive (50% LDH release at 20-35 muM) than the corresponding primary cultures. No difference in sensitivity towards CdMt-1 or CdMt-2 was found for the various cell types tested. To investigate the influence of the molar Cd ratio of CdMt on cytotoxicity, the Cd content of CdMt-1 (2.1 mol Cd/mol Mt) was artificially raised in vitro to 5 mol/mol Mt. Compared with native CdMt, CdMt with a high molar Cd ratio in primary renal cultures showed a 15% increase in LDH release at a Cd concentration of 1500 muM in the medium. In conclusion, exogenous CdMt is far less toxic than CdCl(2) to cell cultures in a serum-free medium. Whereas CdCl(2) in all cases showed dose-dependent Cd accumulation, Cd accumulation due to CdMt exposure in all cell types tested reached a plateau at medium Cd concentrations of 2 muM. The low cellular Cd uptake of CdMt and the corresponding low cytotoxicity supports previously reported results in vivo, showing that the difference in toxicity between CdMt and CdCl(2) is associated with a difference in Cd distribution.

Cryopreservation of precision-cut rat liver slices using a computer-controlled freezer.

Precision-cut liver slices are frequently used to study hepatic toxicity and metabolism of xenobiotics in vitro. Successful cryopreservation techniques will enhance an efficient and economic use of scarcely available (human) liver tissue. For primary hepatocytes, slow freezing has been accepted as the best approach towards successful cryopreservation. For slices, however, no agreement exists on the optimal way of cryopreservation and both slow and fast freezing techniques have been reported. The aim of the present study was to determine the applicability of a computer-controlled slow freezing technique for the cryopreservation of (rat) liver slices. Thus far, this technique has not been described in detail. Our studies confirmed that slow freezing was most successful in the cryopreservation of primary rat hepatocytes. Based on this observation, the slow freezing technique was applied to the cryopreservation of rat liver slices. Directly after thawing, slice viability was between 60 and 100% of fresh values, depending on the parameter determined. However, after additional culturing, slice viability was reduced. This decrease in slice viability was more pronounced in comparison to primary hepatocytes. In conclusion, the slow freezing technique was confirmed to be a successful approach for the cryopreservation of primary rat hepatocytes, and was found to be of limited use for the cryopreservation of rat liver slices.

Mixtures and interactions.

Drinking water can be considered as a complex mixture that consists of tens, hundreds or thousands of chemicals of which the composition is qualitatively and quantitatively not fully known. From a public health point of view it is most relevant to answer the question of whether chemicals in drinking water interact in a way that results in an increased overall response as compared to the sum of the responses to the individual chemicals in the mixture, or indeed in an effect that is simply a summation of the expected effects of the individual chemicals. Present methods for risk assessment of mixtures rely heavily on some form of additivity model, unless data are adequate for a direct risk assessment of the mixture of concern in its entirety. The "dose-addition" concept ("simple similar action") is the most common approach to risk assessment of mixtures and it is applicable over the whole range of exposure levels from low non-toxic to toxic levels when all chemicals in the mixture act in a similar way. However, in toxicity studies at environmentally relevant exposure scenarios the mixtures that meet such conditions are the exception rather than the rule. In that case the "effect addition" model has to be followed assuming "independent joint action". For these compounds now experimental data have indicated that the results at low exposure levels are probably difficult to predict based on response additivity found at higher dose levels. Thus, although the additivity models are mathematically simple, they require assumptions about the mechanisms of action and the high-to-low dose extrapolation. Therefore, theoretical considerations in risk assessment of chemical mixtures should be verified by simple case studies. Up till now, the number of environmentally relevant mixtures to which a direct risk assessment has been devoted is limited. Even if toxicity data on individual compounds are available, we are still facing the immense problem of extrapolation of findings obtained at relatively high exposure concentration in laboratory animals to man being exposed to (much) lower concentrations. Therefore the prioritization of compounds for further research and the extrapolation to low doses should be considered as key issues in the assessment of possible health risks from exposure to chemical mixtures such as drinking water.

Toxicological evaluation of chemical mixtures.

This paper addresses major developments in the safety evaluation of chemical mixtures during the past 15 years, reviews today's state of the art of mixture toxicology, and discusses challenges ahead. Well-thought-out tailor-made mechanistic and empirical designs for studying the toxicity of mixtures have gradually substituted trial-and-error approaches, improving the insight into the testability of joint action and interaction of constituents of mixtures. The acquired knowledge has successfully been used to evaluate the safety of combined exposures and complex mixtures such as, for example, the atmosphere at hazardous waste sites, drinking water disinfection by-products, natural flavouring complexes, and the combined intake of food additives. To consolidate the scientific foundation of mixture toxicology, studies are in progress to revisit the biological concepts and mathematics underlying formulas for low-dose extrapolation and risk assessment of chemical mixtures. Conspicuous developments include the production of new computer programs applicable to mixture research (CombiTool, BioMol, Reaction Network Modelling), the application of functional genomics and proteomics to mixture studies, the use of nano-optochemical sensors for in vivo imaging of physiological processes in cells, and the application of optical sensor micro- and nano-arrays for complex sample analysis. Clearly, the input of theoretical biologists, biomathematicians and bioengineers in mixture toxicology is essential for the development of this challenging branch of toxicology into a scientific subdiscipline of full value.

Use of factorial designs in combination toxicity studies.

The use of factorial designs, in which n chemicals are studied at xn dose levels (x treatment groups), has been put forward as one of the valuable statistical approaches for hazard assessment of chemical mixtures. Very recently a "2(5) study' was presented to describe interactions between the carcinogenic activity of five polycyclic aromatic hydrocarbons and a "5(3) study' was used to identify the non-additive effects of three compounds on developmental toxicity. Full factorial designs, however, lead to very costly experiments and, even if only two dose levels are used, it is not always possible to perform conventional toxicity tests using 2n test groups to identify possible interactions between all chemicals of interest. One way to deal with this problem is the use of fractionated factorial designs. These fractionated designs still identify most of the interactions between the compounds and determine which compounds are important in causing effects, but have the advantage that the number of test groups is manageable. Fractional factorial designs have been shown to be an efficient (i.e. cost-effective) approach to: (a) identify interactive effects between seven trace elements and cadmium accumulation in the body; and (b) describe cases of non-additivity in a mixture of nine chemicals tested in a 4-wk toxicity study in rats.

Statistically designed experiments in a tiered approach to screen mixtures of Fusarium mycotoxins for possible interactions.

This paper presents a test strategy to detect interactive effects between several mycotoxins using a DNA synthesis inhibition assay in L929 cells. The joint action of the Fusarium mycotoxins T-2 toxin (T2), deoxynivalenol (DON), nivalenol (NIV), zearalenone (ZEA) and fumonisin (FB1) was studied in a tiered approach. In the first stage, the mycotoxins were tested either jointly in a five-compound mixture, or individually. At the highest dose level, the mixture showed a clear less than additive action of the mycotoxins, as compared to the effects of the five individual compounds, whereas at lower dose levels the mycotoxins behaved additive. In the second stage, the non-additivity as established in the first experiment was further analyzed with a central composite design to detect interactions between specific mycotoxins in the mixture. This experiment confirmed less than additivity for five of the mixes tested. However, it also revealed four significant synergistic interactions between mycotoxins. Finally, two interactions that were established in stage 2 were further studied in full factorial designs involving two mycotoxins. One of the interactions observed in the central composite design was retrieved whereas the other two-factor interaction was not. It was concluded that several classes of mycotoxins when present simultaneously in a mixture might show interaction. The effect of the mixture cannot be predicted solely on the basis of the effect of the individual compounds.

Comparison of the toxicity of inorganic and liver-incorporated cadmium: a 4-wk feeding study in rats.

The toxicity of cadmium was examined in rats fed diets containing either tissue-incorporated cadmium or cadmium salt for 4 wk. The test diets contained 30 mg cadmium/kg either as cadmium chloride, or as cadmium incorporated in pigs' livers; the control group was fed a diet containing liver from a pig not treated with cadmium. Over 90% of the cadmium present in the pigs' livers was bound to metallothionein. Analysis of the diet and determination of the food consumption revealed that both cadmium-fed groups were exposed to similar dietary cadmium levels. There was no adverse effect on general health or survival. Feeding cadmium resulted in growth retardation and slightly decreased water intake. Moreover, both cadmium-treated groups showed clear signs of anaemia and increased plasma aspartate and alanine aminotransferase activities. For the group fed cadmium chloride, all of these effects were more pronounced than for the group fed cadmium incorporated in liver. Microscopic examination of the liver and kidneys, however, did not reveal any lesion that could be attributed to the cadmium treatment. After exposure to cadmium the spleen showed decreased extramedullary haematopoiesis, an effect that was also more pronounced after feeding of the cadmium chloride than after feeding liver-incorporated cadmium. The differences in the extent of the toxic effects between the inorganic and the tissue-incorporated cadmium were accompanied by differences in the cadmium concentrations in liver and kidneys: the feeding of cadmium incorporated in pigs' livers resulted in about half the accumulation of cadmium in the rats' livers that took place after intake of a diet containing cadmium chloride. In contrast a much less marked difference in cadmium accumulation was observed in the kidneys. Since humans are usually exposed to tissue-incorporated cadmium these findings deserve further investigation, with special attention to the observed difference in tissue accumulation.

Interaction of dietary Ca, P, Mg, Mn, Cu, Fe, Zn and Se with the accumulation and oral toxicity of cadmium in rats.

The toxicity of Cd was examined in rats fed diets containing 30 mg Cd/kg as CdCl2 for 8 wk. The Cd-containing diets were supplemented with various combinations of the minerals Ca, P, Mg, Mn, Cu, Fe, Zn and Se in order to investigate the protective effect of these mineral combinations on Cd accumulation and toxicity. The mineral combinations were chosen such that the effect of the individual components could be analysed. At the end of the 8-wk feeding period, the Cd concentrations in the liver and renal cortex were 13.9 and 19.5 mg/kg body weight, respectively. The feeding of 30 mg Cd/kg diet alone resulted in well known Cd effects, such as growth retardation, slight anaemia, increased plasma transaminase activities and alteration of Fe accumulation. Only supplements that contained extra Fe resulted in a significant protection against Cd accumulation and toxicity. The most pronounced effect was obtained using a supplement of Ca/P, Fe and Zn, which resulted in a 70-80% reduction in Cd accumulation in the liver and kidneys, as well as a reduction in Cd toxicity. The protective effect of the mineral combinations was mainly due to the presence of Fe2+, but in combinations with Ca/P and Zn the effect of Fe was most pronounced. Compared with Fe2+ the protective effect of Fe3+ was significantly lower. Addition of ascorbic acid to Fe in both forms improved the Fe uptake, but consequently did not decrease Cd accumulation. Thus, the mineral status of the diet may have a considerable impact on the accumulation and toxicity of Cd, fed as CdCl2 in laboratory animals. For the risk assessment of Cd intake, special consideration should be given to an adequate intake of Fe.

Experimental designs and risk assessment in combination toxicology: panel discussion.

Advancing our knowledge on the toxicology of combined exposures to chemicals and implementation of this knowledge in guidelines for health risk assessment of such combined exposures are necessities dictated by the simple fact that humans are continuously exposed to a multitude of chemicals. A prerequisite for successful research and fruitful discussions on the toxicology of combined exposures (mixtures of chemicals) is the use of defined terminology implemented by an authoritative international body such as, for example, the International Union of Pure and Applied Chemistry (IUPAC) Toxicology Committee. The extreme complexity of mixture toxicology calls for new research methodologies to study interactive effects, taking into account limited resources. Of these methodologies, statistical designs and mathematical modelling of toxicokinetics and toxicodynamics seem to be most promising. Emphasis should be placed on low-dose modelling and experimental validation. The scientifically sound so-called bottom-up approach should be supplemented with more pragmatic approaches, focusing on selection of the most hazardous chemicals in a mixture and careful consideration of the mode of action and possible interactive effects of these chemicals. Pragmatic approaches may be of particular importance to study and evaluate complex mixtures; after identification of the 'top ten' (most risky) chemicals in the mixture they can be examined and evaluated as a defined (simple) chemical mixture. In setting exposure limits for individual chemicals, the use of an additional safety factor to compensate for potential increased risk due to simultaneous exposure to other chemicals, has no clear scientific justification. The use of such an additional factor is a political rather than a scientific choice.

Safety evaluation of chemical mixtures and combinations of chemical and non-chemical stressors.

Recent developments in hazard identification and risk assessment of chemical mixtures are reviewed. Empirical, descriptive approaches to study and characterize the toxicity of mixtures have dominated during the past two decades, but an increasing number of mechanistic approaches have made their entry into mixture toxicology. A series of empirical studies with simple chemical mixtures in rats is described in some detail because of the important lessons from this work. The development of regulatory guidelines for the toxicological evaluation of chemical mixtures is discussed briefly. Current issues in mixture toxicology include the adverse health effects of ambient air pollution; the application of such modern, sophisticated methodologies as genomics, bioinformatics, and physiologically based pharmacokinetic modeling; and databases for mixture toxicity. Finally, the state of the art of our knowledge on the potential adverse health effects of combined exposures to chemicals and non-chemical stressors (noise, heat/cold, microorganisms, immobilization, restraint, or transportation), research initiatives in these fields, and the development of an indicator for the cumulative health impact of multiple environmental exposures are discussed.

Toxicity of formaldehyde and acrolein mixtures: in vitro studies using nasal epithelial cells.

In vitro studies with human and rat nasal epithelial cells were carried out to investigate the combined toxicity of formaldehyde and acrolein and the role of aldehyde dehydrogenases in this process. These studies showed that the toxic effect of mixtures of aldehydes was additive. In addition, aldehyde dehydrogenases were inhibited by disulfiram and acrolein in S9 incubation but disulfiram did not influence the toxicity in vitro (cell culture). This study does not support the idea that aldehyde dehydrogenases play a major role in the detoxification of exogenous aldehydes.