Cellular Uptake Kinetics of Neutral and Charged Chemicals in in Vitro Assays Measured by Fluorescence Microscopy.

Cellular uptake kinetics are key for understanding time-dependent chemical exposure in in vitro cell assays. Slow cellular uptake kinetics in relation to the total exposure time can considerably reduce the biologically effective dose. In this study, fluorescence microscopy combined with automated image analysis was applied for time-resolved quantification of cellular uptake of 10 neutral, anionic, cationic, and zwitterionic fluorophores in two reporter gene assays. The chemical fluorescence in the medium remained relatively constant during the 24-h assay duration, emphasizing that the proteins and lipids in the fetal bovine serum (FBS) supplemented to the assay medium represent a large reservoir of reversibly bound chemicals with the potential to compensate for chemical depletion by cell uptake, growth, and sorption to well materials. Hence FBS plays a role in stabilizing the cellular dose in a similar way as polymer-based passive dosing, here we term this process as serum-mediated passive dosing (SMPD). Neutral chemicals accumulated in the cells up to 12 times faster than charged chemicals. Increasing medium FBS concentrations accelerated uptake due to FBS-facilitated transport but led to lower cellular concentrations as a result of increased sorption to medium proteins and lipids. In vitro cell exposure results from the interaction of several extra- and intracellular processes, leading to variable and time-dependent exposure between different chemicals and assay setups. The medium FBS plays a crucial role for the thermodynamic equilibria as well as for the cellular uptake kinetics, hence influencing exposure. However, quantification of cellular exposure by an area under the curve (AUC) analysis illustrated that, for the evaluated bioassay setup, current in vitro exposure models that assume instantaneous equilibrium between medium and cells still reflect a realistic exposure because the AUC was typically reduced less than 20% compared to the cellular dose that would result from instantaneous equilibrium.

Potentially harmful elements in house dust from Estarreja, Portugal: characterization and genotoxicity of the bioaccessible fraction.

Due to their behavioral characteristics, young children are vulnerable to the ingestion of indoor dust, often contaminated with chemicals that are potentially harmful. Exposure to potentially harmful elements (PHEs) is currently exacerbated by their widespread use in several industrial, agricultural, domestic and technological applications. PHEs cause adverse health effects on immune and nervous systems and can lead to cancer development via genotoxic mechanisms. The present study is an integrated approach that aims at assessing the genotoxicity of bioaccessible PHEs following ingestion of contaminated house dust. A multidisciplinary methodology associating chemical characterization of five house dust samples, extraction of the bioaccessible PHEs in gastric extracts by the unified BARGE method, determination of the bioaccessible fraction and in vitro genotoxicity of gastric extracts in adenocarcinoma gastric human (AGS) cells was developed. The five gastric extracts induced dose-dependent genotoxicity in AGS cells. Copper (bioaccessible concentration up to 111 mg/kg) was probably the prevalent PHE inducing primary DNA damage (up to 5.1-fold increase in tail DNA at 0.53 g/l of gastric extract). Lead (bioaccessible concentration up to 245 mg/kg) was the most prevalent PHE inducing chromosome-damaging effects (r = 0.55; p < 0.001 for micronucleated cells induction). The association of principal component analysis and Spearman's correlations was decisive to understand the chromosome-damaging properties of the bioaccessible PHEs in AGS cells. This methodology could be used on a larger-scale study to provide useful information for science-based decision-making in regulatory policies, and a better estimation of human exposure and associated health risks.

Bayesian methods for uncertainty factor application for derivation of reference values.

In 2014, the National Research Council (NRC) published Review of EPA's Integrated Risk Information System (IRIS) Process that considers methods EPA uses for developing toxicity criteria for non-carcinogens. These criteria are the Reference Dose (RfD) for oral exposure and Reference Concentration (RfC) for inhalation exposure. The NRC Review suggested using Bayesian methods for application of uncertainty factors (UFs) to adjust the point of departure dose or concentration to a level considered to be without adverse effects for the human population. The NRC foresaw Bayesian methods would be potentially useful for combining toxicity data from disparate sources-high throughput assays, animal testing, and observational epidemiology. UFs represent five distinct areas for which both adjustment and consideration of uncertainty may be needed. NRC suggested UFs could be represented as Bayesian prior distributions, illustrated the use of a log-normal distribution to represent the composite UF, and combined this distribution with a log-normal distribution representing uncertainty in the point of departure (POD) to reflect the overall uncertainty. Here, we explore these suggestions and present a refinement of the methodology suggested by NRC that considers each individual UF as a distribution. From an examination of 24 evaluations from EPA's IRIS program, when individual UFs were represented using this approach, the geometric mean fold change in the value of the RfD or RfC increased from 3 to over 30, depending on the number of individual UFs used and the sophistication of the assessment. We present example calculations and recommendations for implementing the refined NRC methodology.

Human health screening level risk assessments of tertiary-butyl acetate (TBAC): calculated acute and chronic reference concentration (RfC) and Hazard Quotient (HQ) values based on toxicity and exposure scenario evaluations.

A screening level risk assessment has been performed for tertiary-butyl acetate (TBAC) examining its primary uses as a solvent in industrial and consumer products. Hazard quotients (HQ) were developed by merging TBAC animal toxicity and dose-response data with population-level, occupational and consumer exposure scenarios. TBAC has a low order of toxicity following subchronic inhalation exposure, and neurobehavioral changes (hyperactivity) in mice observed immediately after termination of exposure were used as conservative endpoints for derivation of acute and chronic reference concentration (RfC) values. TBAC is not genotoxic but has not been tested for carcinogenicity. However, TBAC is unlikely to be a human carcinogen in that its non-genotoxic metabolic surrogates tertiary-butanol (TBA) and methyl tertiary butyl ether (MTBE) produce only male rat α-2u-globulin-mediated kidney cancer and high-dose specific mouse thyroid tumors, both of which have little qualitative or quantitative relevance to humans. Benchmark dose (BMD)-modeling of the neurobehavioral responses yielded acute and chronic RfC values of 1.5 ppm and 0.3 ppm, respectively. After conservative modeling of general population and near-source occupational and consumer product exposure scenarios, almost all HQs were substantially less than 1. HQs exceeding 1 were limited to consumer use of automotive products and paints in a poorly ventilated garage-sized room (HQ = 313) and occupational exposures in small and large brake shops using no personal protective equipment or ventilation controls (HQs = 3.4-126.6). The screening level risk assessments confirm low human health concerns with most uses of TBAC and indicate that further data-informed refinements can address problematic health/exposure scenarios. The assessments also illustrate how tier-based risk assessments using read-across toxicity information to metabolic surrogates reduce the need for comprehensive animal testing.

Biomarker responses and accumulation of hazardous substances in mussels (Mytilus trossulus) transplanted along a pollution gradient close to an oil terminal in the Gulf of Finland (Baltic Sea).

Baltic Sea blue mussels (Mytilus trossulus) were used as sentinel organisms to detect the biological effects of chemical contamination in the low salinity environment. Mussels naturally adapted to a salinity of ca. 6.0 PSU were caged for 30 days at four sites along an assumed pollution gradient (salinity ca. 4.5 PSU) in the vicinity of Finland's largest oil refinery and harbor Kilpilahti in the Gulf of Finland. Tissue concentrations and accumulation rates of especially organic contaminants (PAHs, PCBs and organotins) were clearly elevated at the innermost coastal stations near the harbor area. Biological effects of contaminant exposure on caged mussels were evaluated by measuring a suite of biomarkers including catalase, glutathione S-transferase, superoxide dismutase, glutathione reductase, lipid peroxidation, acetylcholinesterase activity and lysosomal membrane stability. Mussels transplanted near the harbor area were able to elevate their antioxidant defense in response to environmental contamination. Reduced morphometric condition index and soft tissue growth rate together with increased lipid peroxidation and low lysosomal membrane stability were also observed at the most contaminated site. The results suggest that caging of M. trossulus for four weeks at lower salinity is a feasible method for the detection of environmental pollution also in low salinity areas of the Baltic Sea.

Adverse outcome pathways during early fish development: a conceptual framework for identification of chemical screening and prioritization strategies.

The fish early life-stage (FELS) test guideline (OECD 210 or OCSPP 850.1400) is the most frequently used bioassay for predicting chronic fish toxicity and supporting aquatic ecological risk assessments around the world. For each chemical, the FELS test requires a minimum of 360 fish and 1 to 3 months from test initiation to termination. Although valuable for predicting fish full life-cycle toxicity, FELS tests are labor and resource intensive and, due to an emphasis on apical endpoints, provide little to no information about chemical mode of action. Therefore, the development and implementation of alternative testing strategies for screening and prioritizing chemicals has the potential to reduce the cost and number of animals required for estimating FELS toxicity and, at the same time, provides insights into mechanisms of toxicity. Using three reference chemicals with well-established yet distinct adverse outcome pathways (AOPs) in early life stages of fish, we proposed FELS-specific AOPs as conceptual frameworks for identifying useful chemical screening and prioritization strategies. The reference chemicals selected as case studies were a cardiotoxic aryl hydrocarbon receptor agonist (2,3,7,8-tetrachlorodibenzo-p-dioxin), neurotoxic acetylcholinesterase inhibitor (chlorpyrifos), and narcotic surfactant (linear alkylbenzene sulfonate). Using qualitative descriptions for each chemical during early fish development, we developed generalized AOPs and, based on these examples, proposed a three-tiered testing strategy for screening and prioritizing chemicals for FELS testing. Linked with biologically based concentration-response models, a tiered testing strategy may help reduce the reliance on long-term and costly FELS tests required for assessing the hazard of thousands of chemicals currently in commerce.

Mixtures and their risk assessment in toxicology.

For communities generally and for persons living in the vicinity of waste sites specifically, potential exposures to chemical mixtures are genuine concerns. Such concerns often arise from perceptions of a site's higher than anticipated toxicity due to synergistic interactions among chemicals. This chapter outlines some historical approaches to mixtures risk assessment. It also outlines ATSDR's current approach to toxicity risk assessment. The ATSDR's joint toxicity assessment guidance for chemical mixtures addresses interactions among components of chemical mixtures. The guidance recommends a series of steps that include simple calculations for a systematic analysis of data leading to conclusions regarding any hazards chemical mixtures might pose. These conclusions can, in turn, lead to recommendations such as targeted research to fill data gaps, development of new methods using current science, and health education to raise awareness of residents and health care providers. The chapter also provides examples of future trends in chemical mixtures assessment.

Microarray analysis of the phytoremediation and phytosensing of occupational toxicant naphthalene.

Naphthalene is of global environmental concern because it is assumed to contribute considerably to human cancer risk. Plants are important in removing naphthalene from the atmosphere and soil. However, there remains insufficient knowledge on plant response to this compound. To determine the mechanism of naphthalene uptake and transduction in plants, as well as plant response to this compound, a microarray system was used to analyze gene expression patterns in Arabidopsis thaliana after irrigation with 2.0mM naphthalene. A total of 247 differentially expressed genes were identified as upregulated by naphthalene. These genes might specifically contribute to naphthalene uptake, transformation, conjugation, and compartmentalization in the plant. The potential role of upregulated genes in plant defense to naphthalene and the use of phytosensing for naphthalene detection were also discussed.

Dermal absorption of benzene in occupational settings: estimating flux and applications for risk assessment.

There is growing emphasis in the United States and Europe regarding the quantification of dermal exposures to chemical mixtures and other substances. In this paper, we determine the dermal flux of benzene in neat form, in organic solvents, and in aqueous solutions based on a critical review and analysis of the published literature, and discuss appropriate applications for using benzene dermal absorption data in occupational risk assessment. As part of this effort, we synthesize and analyze data for 77 experimental results taken from 16 studies of benzene skin absorption. We also assess the chemical activity of benzene in simple hydrocarbon solvent mixtures using a thermodynamic modeling software tool. Based on the collective human in vivo, human in vitro, and animal in vitro data sets, we find that the steady-state dermal flux for neat benzene (and benzene-saturated aqueous solutions) ranges from 0.2 to 0.4 mg/(cm²·h). Observed outlier values for some of the animal in vivo data sets are possibly due to the use of test species that have more permeable skin than humans or study conditions that resulted in damage to the skin barrier. Because relatively few dermal absorption studies have been conducted on benzene-containing organic solvents, and available test results may be influenced by study design or vehicle effects, it is not possible to use these data to quantify the dermal flux of benzene for other types of solvent mixtures. However, depending on the application, we describe several potential approaches that can be used to derive a rough approximation of the steady-state benzene dermal flux for these mixtures. Important limitations with respect to quantifying and evaluating the significance of dermal exposures to benzene in occupational settings include a lack of data on (1) factors that affect the dermal uptake of benzene, (2) the dermal flux of benzene for different organic solvent mixtures, (3) meaningful metrics for evaluating the dermal uptake of benzene, (4) steady-state versus non-steady-state dermal flux values for benzene, (5) the effect of skin damage on the dermal flux of benzene, (6) standardized test methods for estimating the dermal flux of benzene, and (7) robust estimates of the evaporation rate of benzene from different liquid vehicles.

Transport pathways for cadmium in the intestine and kidney proximal tubule: focus on the interaction with essential metals.

Cadmium (Cd) is a toxic metal with a propensity to accumulate in the proximal tubules cells (PTC) of the kidney where it can lead to tubular dysfunction and eventually renal failure. Although Cd(2+)-induced nephrotoxicity has been well described there is still uncertainty about how this metal gains entry into these cells to induce toxicity. As a non-essential metal, specific transport proteins for Cd are unlikely to exist. Rather transport proteins/channels used by essential metals (iron, zinc, calcium) are thought to be responsible. When these dietary essential metals are in short supply and deficiencies develop, Cd absorption and toxicity are enhanced. This is primarily due to increased expression of essential metal transport proteins such as divalent metal transporter 1 (DMT1) which can transport Cd in the intestine and enhance toxicity in the kidney. The zinc/bicarbonate sympoters ZIP8 and 14 are expressed at the apical membrane of enterocytes and PTC, and can transport Cd into cells. TRPV5 and 6 are major transporters for calcium in intestine and kidney and may be involved in Cd transport in these locations. Cd in the circulation is bound to proteins such as metallothioneins (MT) which are readily filtered. Two multiligand receptors, megalin and cubulin, reabsorb filtered proteins including albumin and MT by the process of receptor-mediated endocytosis. This review summarises the transport pathways for Cd in the intestine and kidney proximal tubule focusing in particular at how Cd uses essential metal transport processes to gain entry to the circulation and the kidney.

Biotransformation of 2,3,3,3-tetrafluoropropene (HFO-1234yf) in rabbits.

2,3,3,3-Tetrafluoropropene (HFO-1234yf) is a non-ozone-depleting fluorocarbon replacement with a low global warming potential and is developed as refrigerant. Due to lethality observed after high concentration inhalation exposures of HFO-1234yf in a developmental toxicity study with rabbits, the biotransformation of HFO-1234yf was investigated in this species. Female New Zealand White rabbits were exposed to air containing 2000; 10,000; or 50,000 ppm (n=3/concentration) HFO234yf. All inhalation exposures were conducted for 6 h in a dynamic exposure chamber. Animals were individually housed in metabolic cages after the end of the exposures and urines were collected at 12 h intervals for 60 h. For metabolite identification, urine samples were analyzed by (1)H-coupled and (1)H-decoupled (19)F-NMR and by LC/MS-MS or GC/MS. Metabolites were identified by (19)F-NMR chemical shifts, signal multiplicity, (1)H-(19)F coupling constants and by comparison with synthetic reference compounds. In urine samples of rabbits exposed to 2000; 10,000; or 50,000 ppm HFO-1234yf, the predominant metabolite was N-acetyl-S-(3,3,3-trifluoro-2-hydroxypropanyl)-l-cysteine and accounted for app. 48% of total (19)F-NMR signal intensities. S-(3,3,3-Trifluoro-2-hydroxypropanyl)mercaptolactic acid, 3,3,3-trifluoro-1,2-dihydroxypropane, 3,3,3-trifluoro-2-propanol and inorganic fluoride were also present as urinary metabolites. In incubations of rabbit liver S9 fractions containing glutathione, NADPH and HFO-1234yf, 3,3,3-trifluoro-1,2-dihydroxypropane, S-(3,3,3-trifluoro-2-hydroxypropanyl)glutathione, 3,3,3-trifluoro-2-propanol and inorganic fluoride were identified as metabolites of HFO-1234yf by (19)F-NMR. The quantity of recovered metabolites in urine suggest a low extent (<0.1% of dose received) of biotransformation of HFO-1234yf in rabbits, and 95% of all metabolites were excreted within 12 h after the end of the exposures (t(1/2) app. 9.5 h). The obtained results indicate that HFO-1234yf is metabolized in rabbits by a CYP450-mediated epoxidation at low rates and glutathione conjugation of the epoxide. The differences in urinary metabolite patterns between rats and rabbits seen with HFO-1234yf are likely due to species-specific processing of glutathione S-conjugates. Rabbits also show a larger extent of biotransformation of HFO-1234yf.

[Development of new cytoxicity testing systems that include toxicokinetic processes].

Conventional in vitro cytotoxicity tests usually do not include toxicokinetic processes that affect final toxicity in the entire body. To overcome this limitation, we have been developing several types of new toxicity test systems and applying them to evaluate hazardous chemicals or environmental samples. In this review, we described two of these new systems; one is a batch-type gas exposure system based on air-liquid interface culture of lung epithelial cells, and the other is a simple double-layered coculture system incorporating permeation and biotransformation processes occurring in the small intestine. In addition, we introduce our latest approach toward further miniaturization of existing tests, that is, determination of minimum cell number necessary for obtaining physiologically-relevant tissue responses.

Probabilistic dose-response modeling: case study using dichloromethane PBPK model results.

A revised assessment of dichloromethane (DCM) has recently been reported that examines the influence of human genetic polymorphisms on cancer risks using deterministic PBPK and dose-response modeling in the mouse combined with probabilistic PBPK modeling in humans. This assessment utilized Bayesian techniques to optimize kinetic variables in mice and humans with mean values from posterior distributions used in the deterministic modeling in the mouse. To supplement this research, a case study was undertaken to examine the potential impact of probabilistic rather than deterministic PBPK and dose-response modeling in mice on subsequent unit risk factor (URF) determinations. Four separate PBPK cases were examined based on the exposure regimen of the NTP DCM bioassay. These were (a) Same Mouse (single draw of all PBPK inputs for both treatment groups); (b) Correlated BW-Same Inputs (single draw of all PBPK inputs for both treatment groups except for bodyweights (BWs), which were entered as correlated variables); (c) Correlated BW-Different Inputs (separate draws of all PBPK inputs for both treatment groups except that BWs were entered as correlated variables); and (d) Different Mouse (separate draws of all PBPK inputs for both treatment groups). Monte Carlo PBPK inputs reflect posterior distributions from Bayesian calibration in the mouse that had been previously reported. A minimum of 12,500 PBPK iterations were undertaken, in which dose metrics, i.e., mg DCM metabolized by the GST pathway/L tissue/day for lung and liver were determined. For dose-response modeling, these metrics were combined with NTP tumor incidence data that were randomly selected from binomial distributions. Resultant potency factors (0.1/ED(10)) were coupled with probabilistic PBPK modeling in humans that incorporated genetic polymorphisms to derive URFs. Results show that there was relatively little difference, i.e., <10% in central tendency and upper percentile URFs, regardless of the case evaluated. Independent draws of PBPK inputs resulted in the slightly higher URFs. Results were also comparable to corresponding values from the previously reported deterministic mouse PBPK and dose-response modeling approach that used LED(10)s to derive potency factors. This finding indicated that the adjustment from ED(10) to LED(10) in the deterministic approach for DCM compensated for variability resulting from probabilistic PBPK and dose-response modeling in the mouse. Finally, results show a similar degree of variability in DCM risk estimates from a number of different sources including the current effort even though these estimates were developed using very different techniques. Given the variety of different approaches involved, 95th percentile-to-mean risk estimate ratios of 2.1-4.1 represent reasonable bounds on variability estimates regarding probabilistic assessments of DCM.

Gender differences in the disposition and toxicity of metals.

There is increasing evidence that health effects of toxic metals differ in prevalence or are manifested differently in men and women. However, the database is small. The present work aims at evaluating gender differences in the health effects of cadmium, nickel, lead, mercury and arsenic. There is a markedly higher prevalence of nickel-induced allergy and hand eczema in women compared to men, mainly due to differences in exposure. Cadmium retention is generally higher in women than in men, and the severe cadmium-induced Itai-itai disease was mainly a woman's disease. Gender differences in susceptibility at lower exposure are uncertain, but recent data indicate that cadmium has estrogenic effects and affect female offspring. Men generally have higher blood lead levels than women. Lead accumulates in bone and increased endogenous lead exposure has been demonstrated during periods of increased bone turnover, particularly in women in pregnancy and menopause. Lead and mercury, in the form of mercury vapor and methylmercury, are easily transferred from the pregnant women to the fetus. Recent data indicate that boys are more susceptible to neurotoxic effects of lead and methylmercury following exposure early in life, while experimental data suggest that females are more susceptible to immunotoxic effects of lead. Certain gender differences in the biotransformation of arsenic by methylation have been reported, and men seem to be more affected by arsenic-related skin effect than women. Experimental studies indicate major gender differences in arsenic-induced cancer. Obviously, research on gender-related differences in health effects caused by metals needs considerable more focus in the future.

Key scientific issues in the health risk assessment of trichloroethylene.

Trichloroethylene (TCE) is a common environmental contaminant at hazardous waste sites and in ambient and indoor air. Assessing the human health risks of TCE is challenging because of its inherently complex metabolism and toxicity and the widely varying perspectives on a number of critical scientific issues. Because of this complexity, the U.S. Environmental Protection Agency (EPA) drew upon scientific input and expertise from a wide range of groups and individuals in developing its 2001 draft health risk assessment of TCE. This scientific outreach, which was aimed at engaging a diversity of perspectives rather than developing consensus, culminated in 2000 with 16 state-of-the-science articles published together as an Environmental Health Perspectives supplement. Since that time, a substantial amount of new scientific research has been published that is relevant to assessing TCE health risks. Moreover, a number of difficult or controversial scientific issues remain unresolved and are the subject of a scientific consultation with the National Academy of Sciences coordinated by the White House Office of Science and Technology Policy and co-sponsored by a number of federal agencies, including the U.S. EPA. The articles included in this mini-monograph provide a scientific update on the most prominent of these issues: the pharmacokinetics of TCE and its metabolites, mode(s) of action and effects of TCE metabolites, the role of peroxisome proliferator-activated receptor in TCE toxicity, and TCE cancer epidemiology.

Issues in the pharmacokinetics of trichloroethylene and its metabolites.

Much progress has been made in understanding the complex pharmacokinetics of trichloroethylene (TCE) . Qualitatively, it is clear that TCE is metabolized to multiple metabolites either locally or into systemic circulation. Many of these metabolites are thought to have toxicologic importance. In addition, efforts to develop physiologically based pharmacokinetic (PBPK) models have led to a better quantitative assessment of the dosimetry of TCE and several of its metabolites. As part of a mini-monograph on key issues in the health risk assessment of TCE, this article is a review of a number of the current scientific issues in TCE pharmacokinetics and recent PBPK modeling efforts with a focus on literature published since 2000. Particular attention is paid to factors affecting PBPK modeling for application to risk assessment. Recent TCE PBPK modeling efforts, coupled with methodologic advances in characterizing uncertainty and variability, suggest that rigorous application of PBPK modeling to TCE risk assessment appears feasible at least for TCE and its major oxidative metabolites trichloroacetic acid and trichloroethanol. However, a number of basic structural hypotheses such as enterohepatic recirculation, plasma binding, and flow- or diffusion-limited treatment of tissue distribution require additional evaluation and analysis. Moreover, there are a number of metabolites of potential toxicologic interest, such as chloral, dichloroacetic acid, and those derived from glutathione conjugation, for which reliable pharmacokinetic data is sparse because of analytical difficulties or low concentrations in systemic circulation. It will be a challenge to develop reliable dosimetry for such cases.

Integrated testing strategies for use in the EU REACH system.

Integrated testing strategies have been proposed to facilitate the process of chemicals risk assessment to fulfil the requirements of the proposed EU REACH system. Here, we present individual, decision-tree style, strategies for the eleven major toxicity endpoints of the REACH system, including human health effects and ecotoxicity. These strategies make maximum use of non-animal approaches to hazard identification, before resorting to traditional animal test methods. Each scheme: a) comprises a mixture of validated and non-validated assays (distinguished in the schemes); and b) decision points at key stages to allow the cessation of further testing, should it be possible to use the available information to classify and label and/or undertake risk assessment. The rationale and scientific justification for each of the schemes, with respect to the validation status of the tests involved and their individual advantages and limitations, will be discussed in detail in a series of future publications.

Are there sex and gender differences in acute exposure to chemicals in the same setting?

We have little understanding of the influence that sex and gender may have on exposure to and measurement of occupational chemicals. If men and women are in the same physical environment, whether that be an occupational or an environmental setting, researchers need to question whether their acute exposure, as measured by administered and/or biologically effective dose, is the same. Not doing so may result in incorrect inferences being made about the risks associated with that exposure. Three critical questions arise specifically, do men and women differ in (1) their personal environments (immediate physical environments and personal attributes), (2) their absorption of the substance across the various biological barriers, and (3) the amount of active substance that reaches the target sites? Both contextual (e.g., smoking habits, diet, use of personal care products and jewellery, hobbies, stress, and use of medications) and biological (e.g., endocrine status) factors should be considered in answering these questions. Examples from the literature are provided to show that, depending on the chemical compound, there may be sex and gender differences in exposure to chemicals which can be manifested in sex differences in absorption, distribution, metabolism, storage, and excretion. An argument is developed to support the need to make information available, such as pharmacokinetic modeling studies in both men and women including appropriate age groups representing the spectrum of life stages and reproductive status.

Multimethod characterization of the interaction of aluminum ion with alpha-ketoglutaric acid in acidic aqueous solutions.

It has recently been reported that aluminum plays a very important role in reducing the activity of Krebs-cycle enzymes and glutamate dehydrogenase in rat brain homogenate. Therefore, it is necessary to identify the aluminum binding ability with the pivotal substrate alpha-ketoglutarate in biological systems. The interactions of aluminum with alpha-ketoglutarate were studied with pH-potentiometry, cyclic voltammetry, UV-vis, 1H, 27Al-NMR and Raman spectra multi-analytical techniques in acidic aqueous solution to measure the stoichiometries and stability constants of the complexes and its keto-enol tautomerism. The alpha-ketoglutarate was found to bind Al in a bidentate manner at the carboxylate and carbonyl moieties. The mononuclear 1:1 (AlLH(-1), AIL+, AlHL2+) and 2:1 (AlL2-, AlL2H(-2)3-) species, and dinuclear 2:1 (Al2L4+) species were found in acidic aqueous solution. Meanwhile, Al can promote alpha-KG tautomerize to its enolic-structure compounds in solutions. These findings may help to further understand the influence of Al on GDH enzyme reactions in biological systems.