An updated lymphohematopoietic and bladder cancers risk evaluation for occupational and environmental exposures to 1,3-butadiene.

EPA designated 1,3-butadiene (BD) as a high priority chemical in December 2019 and is presently performing an evaluation under the Toxic Substances Control Act (TSCA). EPA's cancer dose-response assessment for BD was published in 2002 and was primarily based on a study on workers exposed to BD in the North American synthetic Styrene-Butadiene Rubber (SBR) Industry developed by the University of Alabama at Birmingham (UAB). EPA relied upon a Poisson regression of leukemia mortality data from this cohort (hereinafter referred to as the SBR study) to estimate the cancer potency of BD. At the time, the SBR cohort included more than 15,000 male workers that were followed up through 1991. The SBR cohort has undergone multiple updates over the past two decades. Most recently, Sathiakumar et al. (2021a, b) published an update, with 18 more years of follow up in addition to approximately 5,000 female workers and updated exposure concentration estimates. Recent EPA assessments (e.g., for ethylene oxide, USEPA 2016) based on epidemiological studies use Cox proportional hazards models because they offer better control of the effect of age in cancer development and are less restrictive than Poisson regression models. Here, we develop exposure-response models using standard Cox proportional hazards regression. We explore the relationship between six endpoints (all leukemia, lymphoid leukemia, myeloid leukemia, multiple myeloma, non-Hodgkin's lymphoma, and bladder cancer) and exposures to BD using the most recent exposure metrics and the most recent update of the SBR study. After adjusting for statistically significant covariates, an upper 95% confidence level on the cancer potency based on leukemia derived herein is 0.000086 per ppm, which is approximately 1,000-fold less than EPA's (2002) estimate of 0.08 per ppm and about 10-fold less than TCEQ's (2008) estimate of 0.0011 per ppm.

Mode of action and dose-response framework analysis for receptor-mediated toxicity: The aryl hydrocarbon receptor as a case study.

Dioxins and dioxin-like compounds are tumor promoters that cause liver cancer in rats and mice. The aryl hydrocarbon receptor (AHR) has been implicated as a key component in this tumor promotion response. Despite extensive knowledge of the toxicology of dioxins, no mode of action (MOA) hypothesis for their tumorigenicity has been formally documented using the Human Relevance MOA framework developed by the International Programme on Chemical Safety (IPCS). To address this information gap, an expert panel was convened as part of a workshop on receptor-mediated liver tumorigenicity. Liver tumors induced by ligands of the AHR were assessed using data for dioxins and related chemicals as a case study. The panel proposed a MOA beginning with sustained AHR activation, eventually leading to liver tumors via a number of other processes, including increased cell proliferation of previously initiated altered hepatic foci, inhibition of intrafocal apoptosis and proliferation of oval cells. These processes have been identified and grouped as three key events within the hepatocarcinogenic MOA: (1) sustained AHR activation, (2) alterations in cellular growth and homeostasis and (3) pre-neoplastic tissue changes. These key events were identified through application of the Bradford-Hill considerations in terms of both their necessity for the apical event/adverse outcome and their human relevance. The panel identified data supporting the identification and dose-response behavior of key events, alteration of the dose-response by numerous modulating factors and data gaps that potentially impact the MOA. The current effort of applying the systematic frameworks for identifying key events and assessing human relevance to the AHR activation in the tumorigenicity of dioxins and related chemicals is novel at this time. The results should help direct future regulatory efforts and research activities aimed at better understanding the potential human cancer risks associated with dioxin exposure.

Mechanism-based common reactivity pattern (COREPA) modelling of aryl hydrocarbon receptor binding affinity.

The aryl hydrocarbon receptor is a ligand-activated transcription factor responsive to both natural and synthetic environmental compounds, with the most potent agonist being 2,3,7,8-tetrachlotrodibenzo-p-dioxin. The aim of this work was to develop a categorical COmmon REactivity PAttern (COREPA)-based structure-activity relationship model for predicting aryl hydrocarbon receptor ligands within different binding ranges. The COREPA analysis suggested two different binding mechanisms called dioxin- and biphenyl-like, respectively. The dioxin-like model predicts a mechanism that requires a favourable interaction with a receptor nucleophilic site in the central part of the ligand and with electrophilic sites at both sides of the principal molecular axis, whereas the biphenyl-like model predicted a stacking-type interaction with the aryl hydrocarbon receptor allowing electron charge transfer from the receptor to the ligand. The current model was also adjusted to predict agonistic/antagonistic properties of chemicals. The mechanism of antagonistic properties was related to the possibility that these chemicals have a localized negative charge at the molecule's axis and ultimately bind with the receptor surface through the electron-donating properties of electron-rich groups. The categorization of chemicals as agonists/antagonists was found to correlate with their gene expression. The highest increase in gene expression was elicited by strong agonists, followed by weak agonists producing lower increases in gene expression, whereas all antagonists (and non-aryl hydrocarbon receptor binders) were found to have no effect on gene expression. However, this relationship was found to be quantitative for the chemicals populating the areas with extreme gene expression values only, leaving a wide fuzzy area where the quantitative relationship was unclear. The total concordance of the derived aryl hydrocarbon receptor binding categorical structure-activity relationship model was 82% whereas the Pearson's coefficient was 0.88.

A pilot study of oral bioavailability of dioxins and furans from contaminated soils: Impact of differential hepatic enzyme activity and species differences.

An in vivo pilot study of the oral bioavailability of polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) in two soils with distinct congener profiles (one dominated by PCDDs, the other by PCDFs) was conducted in rats and juvenile swine. The pilot study revealed potential confounding of relative bioavailability estimates compared to bioavailability in spiked corn oil gavage for tetrachlorodibenzofuran (TCDF) in the rat study due to differential EROD induction between groups receiving soil and those receiving spiked control PCDDs/PCDFs. A follow-up study in rats with the furan-contaminated soil was then conducted with reductions in the spiked control doses to 20%, 50% and 80% of the soil-feed dose in order to bracket hepatic enzyme induction levels in the soil group. When hepatic enzyme induction was matched between the soil and spiked control groups, the apparent relative bioavailability for TCDF was reduced significantly. Overall, after controlling for hepatic enzyme induction, estimates of relative bioavailability in rats and swine differed for the two soils. In the rat study, the relative bioavailability of the two soils were approximately 37% and 60% compared to corn oil administration for the PCDD- and PCDF- dominated soils, respectively, on a TEQ basis. In swine, both soils demonstrated relative bioavailability between 20% and 25% compared to administration in corn oil. These species differences and experimental design issues, such as controlling for differential enzyme induction between corn oil and soil-feed animals in a bioavailability study, are relevant to risk assessment efforts where relative bioavailability inputs are important for theoretical exposure and risk characterization.

Sex ratio of the offspring of Sprague-Dawley rats exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in utero and lactationally in a three-generation study.

Reports of a decreased male/female sex ratio in children born to males exposed to TCDD in Seveso, Italy, at a young age have sparked examinations of this endpoint in other populations exposed to TCDD or related compounds. Overall, the male/female sex ratio results reported in these studies, with slightly different age-exposed male populations, have shown mixed results. Experimental studies of the effects of in utero exposure to TCDD in laboratory animals have reported no effect on the f(1) sex ratio and mixed results for the sex ratio of the f(2) generation. In order to better understand the potential effects of TCDD on second generation sex ratio, we retrieved archived data from a comprehensive three-generation feeding study of TCDD in rats that was conducted and published in the 1970s, but which did not publish data on sex ratio of the offspring [Murray, F.J., Smith, F.A., Nitschke, K.D., Humiston, C.G., Kociba, R.J., Schwetz, B.A., 1979. Three-generation reproduction study of rats given 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the diet. Toxicol. Appl. Pharmacol. 50, 241-252]. A re-examination of the original Murray et al. data found no statistically significant treatment-related changes in postnatal day 1 sex ratio in any generation of treated animals, consistent with one other relatively large study reporting on this endpoint. We discuss mechanistic data underlying a potential effect of TCDD on this endpoint. We conclude that the inconsistency in findings on sex ratio of the offspring of male rats exposed to TCDD in utero is likely due to random variation associated with a relatively small sample size, although differences between studies in strain of rat, dose regimen, and day of ascertainment of sex ratio cannot be ruled out.

PCBs and neurodevelopmental effects in Michigan children: an evaluation of exposure and dose characterization.

Despite the fact that PCB levels in the general environment have continued to decline over the past decade, concern for potential neurodevelopmental deficits from in utero exposure to these compounds remains unabated. In fact, some regulatory and scientific bodies have concluded that the evidence suggesting that prenatal exposure to PCBs may lead to neurodevelopmental deficits is one of the greatest public health concerns surrounding PCBs. The primary basis for the concern that low-level in utero exposure to PCBs causes neurodevelopmental deficits in children is a series of reports on a cohort of Michigan children presumably exposed to PCBs as a result of their mother's consumption of Great Lakes fish. These children, known collectively as the Jacobson cohort, have been followed from birth to 11 years of age. The investigators following these children concluded that they have demonstrated persistent neurodevelopmental effects in this cohort attributable solely to PCBs. However, a detailed analysis of the cohort's exposure characterization, particularly in the initial reports, reveals considerable uncertainty as to the actual exposure status of mothers characterized as "fish eaters" and their offspring. Failure to adequately characterize the PCB exposure of these mothers, or their children, precludes any causal association between in utero exposure to PCBs and neurodevelopmental deficits.

Primaquine-induced hemolytic anemia: formation and hemotoxicity of the arylhydroxylamine metabolite 6-methoxy-8-hydroxylaminoquinoline.

Primaquine is an important antimalarial agent because of its activity against exoerythrocytic forms of Plasmodium spp. However, methemoglobinemia and hemolytic anemia are dose-limiting side effects of primaquine therapy that limit its efficacy. These hemotoxicities are thought to be mediated by metabolites; however, the identity of the toxic species has remained unclear. Since N-hydroxy metabolites are known to mediate the hemotoxicity of several arylamines, the present studies were undertaken to determine whether 6-methoxy-8-aminoquinoline (6-MAQ), a known human metabolite of primaquine, could undergo N-hydroxylation to form a hemotoxic metabolite. When 6-MAQ was incubated with rat and human liver microsomes, a single metabolite was detected by high performance liquid chromatography (HPLC) with electrochemical detection. This metabolite was identified as 6-methoxy-8-hydroxylaminoquinoline (MAQ-NOH) by HPLC and mass spectral analyses. As measured by decreased survival of (51)Cr-labeled erythrocytes in rats, MAQ-NOH was hemolytic in vivo. Furthermore, in vitro exposure of (51)Cr-labeled erythrocytes to MAQ-NOH caused a concentration-dependent decrease in erythrocyte survival (EC(50) of 350 microM) when the exposed cells were returned to the circulation of isologous rats. MAQ-NOH also induced the formation of methemoglobin when incubated with suspensions of rat erythrocytes. These data indicate that 6-MAQ can be metabolized to MAQ-NOH by both rat and human liver microsomes and that MAQ-NOH has the requisite properties to be a hemotoxic metabolite of primaquine. The contribution of MAQ-NOH to the hemotoxicity of primaquine in vivo remains to be assessed.

An evaluation of modeled benzene exposure and dose estimates published in the Chinese-National Cancer Institute collaborative epidemiology studies.

Risk estimates and cause and effect determinations are directly dependent on exposure and dose-response relationships. Recently, relative risks and excess cancer mortality attributed to occupational benzene exposure have been published in collaborative studies conducted by Chinese investigators and scientists from the National Cancer Institute. The results of these studies suggest increased risk of acute nonlymphocytic leukemia at relatively low benzene concentrations and associations with cancers not previously associated with benzene exposure. These studies are potentially important due to their size and potential to more thoroughly investigate the link between benzene exposure and cancer. However, there are questions concerning the validity of exposure and dose estimates supporting relative risk characterizations in these studies. Apparent discrepancies between modeled exposure and dose estimates and sources of actual measured exposure information and clinical markers of benzene toxicity raise serious concerns questioning the reliability of relative risk and cancer associations stated in these studies.

Dapsone-induced hemolytic anemia: effect of dapsone hydroxylamine on sulfhydryl status, membrane skeletal proteins and morphology of human and rat erythrocytes.

Dapsone hydroxylamine is a direct-acting hemolytic agent responsible for dapsone-induced hemolytic anemia in the rat. In the present study, we compared the responsiveness of rat and human red cells to dapsone hydroxylamine-induced cellular changes. Dapsone hydroxylamine induced a rapid and concentration-dependent loss of erythrocytic reduced glutathione content with a concomitant increase in protein-glutathione mixed disulfide formation in both human and rat red cell suspensions. However, the rate of mixed disulfide formation in human cells was considerably slower than that in rat cells and was preceded by a transient increase in oxidized glutathione (glutathione disulfide) formation. Sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting analysis of membrane ghosts from human red cells revealed changes in skeletal proteins that in general were similar to those observed with rat cells, including a loss of protein band 2.1 and the appearance of membrane-bound hemoglobin. Notable differences were the resistance to loss of band 4.2 and a considerably higher amount of protein aggregation in human ghosts. Although the morphology of human red cells was altered, the incidence and degree of change were considerably less than those of rat red cells. Furthermore, the concentration of dapsone hydroxylamine required to induce damage in human red cells (175-750 microM) was significantly higher than that required for rat red cells (50-175 microM), suggesting that human cells are probably less sensitive than rat cells to dapsone hydroxylamine-induced oxidative damage.

Dapsone-induced hemolytic anemia: role of glucose-6-phosphate dehydrogenase in the hemolytic response of rat erythrocytes to N-hydroxydapsone.

Individuals deficient in erythrocytic glucose-6-phosphate dehydrogenase (G6PD) show about a 2-fold increase in sensitivity toward dapsone-induced hemolytic anemia. Rat studies have shown that the hemolytic activity of dapsone resides in its N-hydroxy metabolites; exposure of rat red cells to N-hydroxy-dapsone in vitro followed by readministration to isologous rats results in premature splenic sequestration of the damaged cells. This study examines the ability of the steroid, epiandrosterone, to inhibit rat red cell G6PD and the effect of such inhibition on the susceptibility of rat red cells to N-hydroxydapsone hemolytic activity. Epiandrosterone was found to inhibit rat red cell G6PD uncompetitively and to suppress red cell hexose monophosphate shunt activity by more than 95%. Epiandrosterone suppression of rat red cell G6PD activity resulted in about a 2-fold increase in sensitivity of the rat cells to N-hydroxydapsone hemolytic activity, and a modest but significant increase in depletion of red cell glutathione. In contrast, suppression of rat red cell catalase activity by aminotriazole had no effect on the hemotoxicity of N-hydroxydapsone. Epiandrosterone appears to be a useful tool to explore the mechanism by which G6PD deficiency enhances susceptibility to hemolytic drugs.

Immunomodulatory effects of procainamide metabolites: their implications in drug-related lupus.

Evidence suggests that N-oxidized metabolites of procainamide may be responsible for the development of lupus-like symptoms associated with procainamide therapy. The human hepatic microsomal metabolism of procainamide has been previously reported to result in formation of the N-hydroxylamine derivative of procainamide (procainamide hydroxylamine [PAHA]). The objective of this study was to examine the effects of PAHA on human lymphocytes and adherent cells (monocytes and macrophages). When incubated with lymphocytes in whole blood, PAHA enhanced the response to mitogen and immunoglobulin secretion at lower concentrations (less than or equal to 4 mumol/L) but suppressed these functions at higher concentrations. The cytotoxic effects were nonselective for T lymphocytes and B lymphocytes and appeared to involve an interaction between PAHA and hemoglobin. When erythrocytes were removed or when hemoglobin was converted to carboxyhemoglobin, the suppressive effects of PAHA on lymphocytes were reduced. PAHA stimulated interleukin-1 production by adherent cells at 25 mumol/L but had no effect at lower concentrations. Superoxide anion release was unaffected by PAHA in "resting" adherent cells. Pretreatment with PAHA (2 mumol/L) diminished superoxide release in response to stimulation by phorbol myristate acetate (PMA) or latex bead phagocytosis but augmented superoxide release when coincubated with PMA or latex. These observations indicate that PAHA produces complex, concentration-dependent interactions with human immunoregulatory cells, and they suggest that the effects of PAHA on lymphocyte function may result from the further oxidation of PAHA by hemoglobin, perhaps to the nitroso form.

Procainamide hydroxylamine lymphocyte toxicity--I. Evidence for participation by hemoglobin.

A number of lines of evidence suggest that the lupus-like symptoms associated with procainamide therapy may be caused by products of metabolic N-oxidation. In the present study, the perfusion of the isolated rat liver with a hemoglobin-free solution containing procainamide (100 microM) resulted in the rapid appearance of the N-oxidation metabolite procainamide hydroxylamine in the perfusate. Addition of procainamide hydroxylamine in vitro to whole rat blood (1-40 microM) resulted in a concentration-dependent loss of proliferative response among mononuclear cells isolated from the treated blood and cultured with mitogens (phytohemagglutinin, PHA-P: concanavalin A, Con A; and pokeweed mitogen, PWM), as well as a loss of viability. Similar effects on lymphocyte mitogen responsiveness were observed when procainamide hydroxylamine (1-40 microM) was added to rat whole splenic cell populations. Carbon monoxide or ascorbic acid pretreatment inhibited the toxicity of procainamide hydroxylamine to lymphocytes in whole blood, but only carbon monoxide pretreatment inhibited procainamide hydroxylamine-induced methemoglobin formation. These observations are consistent with the participation of hemoglobin in a redox cycle with procainamide hydroxylamine, generating products which are primarily responsible for its cytotoxicity in blood.

The formation of procainamide hydroxylamine by rat and human liver microsomes.

A method is described, using HPLC and electrochemical detection, which permits the direct quantitation of procainamide hydroxylamine. Procainamide hydroxylamine was formed from procainamide by hepatic microsomes from both rat and human, with rat microsomes showing higher apparent formation rates. The apparent Km for formation of procainamide hydroxylamine was 0.044 mM for rat liver microsomes, with an apparent Vmax of 2.81 nmol/min/mg of protein. Estimates of Km from three human microsomal samples were 6.29, 2.89, and 6.88 mM. Vmax estimates were 0.31, 0.74, and 0.74 nmol/min/mg of protein, respectively, roughly an order of magnitude less than that observed for the rat. Microsomal formation in both species was inhibited by boiling the microsomes, eliminating NADPH from the incubation system, by preincubation with SKF 525A, cimetidine, or n-octylamine, or by gassing the microsomal incubation mixture with carbon monoxide. These observations suggest that procainamide hydroxylamine formation is cytochrome P-450 mediated. Procainamide hydroxylamine could not be detected in the blood of rats treated with a single dose of procainamide, 100 mg/kg, po. One potential reason for the inability to detect this metabolite in blood is indicated by the rapid disappearance in vitro of procainamide hydroxylamine added to whole blood. Most of this disappearance appears to be due to an interaction with hemoglobin.