Microcystins (MCs) are a large group of heptapeptide cyanobacterial toxins commonly produced in harmful algal blooms (HABs) and associated with adverse health effects in wildlife, livestock, pets, and humans. MC chemical standards are extracted from cyanobacteria biomass rather than produced synthetically and are used in water assessment methods and toxicological studies. MC standards are generally supplied in less than 1 mg quantities, and verification of the mass can only be accomplished by analytical chemistry methods using a certified reference of the specific MC for comparison. Analytical quantification of MCs in environmental samples and toxicology studies using accurate doses of test chemicals administered to experimental animals rely on the availability and accuracy of chemical standards. To check the accuracy and purity of available standards, seven individual microcystin-LR (MCLR) standards were purchased from separate commercial vendors and analyzed to determine the actual mass supplied and identify the presence of potential contaminants. To determine the effect of varying toxin mass in toxicological studies, each MCLR standard was administered to CD-1 mice in doses based on mass purchased, by a single 40 µg/kg intraperitoneal injection. The measured mass purchased varied from the vendor label mass by more than 35% for two of the seven MCLR standards. Contaminants, including trifluoroacetic acid (TFA), were identified in four of the seven samples. Comparative in vivo hepatotoxicity between vendor samples closely reflected the actual amount of MCLR present in each standard and demonstrated the toxicological impact of varying cyanotoxin mass.

Although the health benefits of swimming are well-documented,health effects such as asthma and bladder cancer are linked to disinfection by-products(DBPs)in pool water.DBPs are formed from the reaction of disinfectants such as chlorine(Cl)or bromine(Br)with organics in the water.Our previous study(Daiber et al.,Environ.Sci.Technol.50,6652;2016)found correlations between the concentrations of classes of DBPs and the mu-tagenic potencies of waters from chlorinated or brominated swimming pools and spas.We extended this study by identifying significantly different concentrations of 21 individ-ual DBPs in brominated or chlorinated pool and spa waters as well as identifying which DBPs and additional DBP classes were most associated with the mutagenicity of these wa-ters.Using data from our previous study,we found that among 21 DBPs analyzed in 21 pool and spa waters,the concentration of bromoacetic acid was significantly higher in Br-waters versus Cl-waters,whereas the concentration of trichloroacetic acid was significantly higher in Cl-waters.Five Br-DBPs(tribromomethane,dibromochloroacetic acid,dibromoace-tonitrile,bromoacetic acid,and tribromoacetic acid)had significantly higher concentra-tions in Br-spa versus Cl-spa waters.Cl-pools had significantly higher concentrations of Cl-DBPs(trichloroacetaldehyde,trichloromethane,dichloroacetic acid,and chloroacetic acid),whereas Br-pools had significantly higher concentrations of Br-DBPs(tribromomethane,di-bromoacetic acid,dibromoacetonitrile,and tribromoacetic acid).The concentrations of the sum of all 4 trihalomethanes,all 11 Br-DBPs,and all 5 nitrogen-containing DBPs were each significantly higher in brominated than in chlorinated pools and spas.The 8 Br-DBPs were the only DBPs whose individual concentrations were significantly correlated with the muta-genic potencies of the pool and spa waters.These results,along with those from our earlier study,highlight the importance of Br-DBPs in the mutagenicity of these recreational waters.

Microcystins are common freshwater cyanobacterial toxins that affect liver function. The toxicities of five microcystin congeners (microcystin-LA (MCLA), MCLR, MCLY, MCRR, and MCYR) commonly observed in harmful algal blooms (HABs) were evaluated in BALB/c mice after a single oral administration of doses ranging from those that were no observed adverse effect levels (NOAELs) to lowest observed adverse effect levels (LOAELs). Animals were monitored for changes in behavior and appearance, and euthanized 24 h after dosing. Test endpoints included clinical changes, necropsy observations, and serum indicators of hepatic toxicity and general homeostasis. Doses were 0.5–7 mg/kg MCLA, 0.5–11 mg/kg MCLR, 1–7 mg/kg MCLY, 7–22 mg/kg MCRR, and 3–11 mg/kg MCYR. MCLA at 3 mg/kg elevated liver/body weight ratio and liver score, ALT, AST, and GLDH, indicating hepatic toxicity, reduced serum glucose and highly elevated total serum bilirubin. MCLR and MCLY induced similar effects with LOAELs of 5 mg/kg, although a greater extent and severity of effects were observed in MCLR animals. MCRR exposure at 22 mg/kg was associated with reduced serum glucose. MCYR induced scattered liver effects at 7 mg/kg and reduced serum glucose levels at 5 mg/kg. The results indicate significant differences in congener-induced toxicity after microcystin exposure.

Short-term biomarkers of toxicity have an increasingly important role in the screening and prioritization of new chemicals. In this study, we examined early indicators of liver toxicity for three reference organophosphate (OP) chemicals, which are among the most widely used insecticides in the world. The OP methidathion was previously shown to increase the incidence of liver toxicity, including hepatocellular tumors, in male mice. To provide insights into the adverse outcome pathway (AOP) that underlies these tumors, effects of methidathion in the male mouse liver were examined after 7 and 28 day exposures and compared to those of two other OPs that either do not increase (fenthion) or possibly suppress liver cancer (parathion) in mice. None of the chemicals caused increases in liver weight/body weight or histopathological changes in the liver. Parathion decreased liver cell proliferation after 7 and 28 days while the other chemicals had no effects. There was no evidence for hepatotoxicity in any of the treatment groups. Full-genome microarray analysis of the livers from the 7 and 28 day treatments demonstrated that methidathion and fenthion regulated a large number of overlapping genes, while parathion regulated a unique set of genes. Examination of cytochrome P450 enzyme activities and use of predictive gene expression biomarkers found no consistent evidence for activation of AhR, CAR, PXR, or PPARα. Parathion suppressed the male-specific gene expression pattern through STAT5b, similar to genetic and dietary conditions that decrease liver tumor incidence in mice. Overall, these findings indicate that methidathion causes liver cancer by a mechanism that does not involve common mechanisms of liver cancer induction.

Per- and polyfluoroalkyl substances (PFAS) are organic chemicals with wide industrial and consumer uses. They are found ubiquitously at low levels in the environment and are detectable in humans and wildlife. Perfluorobutane Sulfonate (PFBS) is a short-chained PFAS used to replace perfluorooctane sulfonate in commerce. In general, the rate of clearance for the short-chained PFAS is faster than that for the long-chained congeners. This study evaluated the pharmacokinetic properties of PFBS and its hepatic transcriptional responses in CD-1 mice. Males and females were given PFBS by oral gavage at 30 or 300 mg/kg; controls received 0.5 % Tween-20 vehicle. Trunk blood was collected at 0.5, 1, 2, 4, 8, 16 and 24 h thereafter; liver and kidney were also harvested. Serum and tissue concentrations of PFBS were determined by HPLC-MS-MS. Expression of several hepatic nuclear receptor target genes was determined by qPCR. The half-life of PFBS was estimated as 5.8h in the males and 4.5 h in the females. Tmax was reached within 1 - 2 h. Volume of distribution was similar between the two sexes (0.32 - 0.40 L/kg). The rate of PFBS clearance was linear with exposure doses. Within 24 h, serum PFBS declined to less than 5 % of Cmax. PFBS was detected in liver or kidney, although tissue levels of the chemical were only a fraction of those in serum. At 24 h after administration of 300 mg/kg PFBS, elevated expression of several hepatic genes targeted for PPAR alpha, PPARy, and PXR but not by AhR, LXR or CAR was observed, with responses indistinguishable between males and females. Little to no transcriptional response was seen with the 30 mg/kg dose. The short serum half-lives of PFBS (4 - 5 h) in mice were comparable to those reported in rats. Although detection of PFBS in liver was low compared to that in serum even at the 300 mg/kg dose, the tissue level was sufficient to activate several hepatic nuclear receptors, which may represent an acute response to the chemical at a high dose.

BACKGROUND: Per- and polyfluoroalkyl substances (PFAS) are a diverse class of industrial chemicals with widespread environmental occurrence. Exposure to long-chain PFAS is associated with developmental toxicity, prompting their replacement with short-chain and fluoroether compounds. There is growing public concern over the safety of replacement PFAS. OBJECTIVE: We aimed to group PFAS based on shared toxicity phenotypes. METHODS: Zebrafish were developmentally exposed to 4,8-dioxa-3H-perfluorononanoate (ADONA), perfluoro-2-propoxypropanoic acid (GenX Free Acid), perfluoro-3,6-dioxa-4-methyl-7-octene-1-sulfonic acid (PFESA1), perfluorohexanesulfonic acid (PFHxS), perfluorohexanoic acid (PFHxA), perfluoro-n-octanoic acid (PFOA), perfluorooctanesulfonic acid (PFOS), or 0.4% dimethyl sulfoxide (DMSO) daily from 0-5 d post fenilization (dpf). At 6 dpf, developmental toxicity and developmental neurotoxicity assays were performed, and targeted analytical chemistry was used to measure media and tissue doses. To test whether aliphatic sulfonic acid PFAS cause the same toxicity phenotypes, perfluorobutanesulfonic acid (PFBS; 4-carbon), perfluoropentanesulfonic acid (PFPeS; 5-carbon), PFHxS (6-carbon), perfluoroheptanesulfonic acid (PFHpS; 7-carbon), and PFOS (8-carbon) were evaluated. RESULTS: PFHxS or PFOS exposure caused failed swim bladder inflation, abnormal ventroflexion of the tail, and hyperactivity at nonteratogenic concentrations. Exposure to PFHxA resulted in a unique hyperactivity signature. ADONA, PFESA1, or PFOA exposure resulted in detectable levels of parent compound in larval tissue but yielded negative toxicity results. GenX was unstable in DMSO, but stable and negative for toxicity when diluted in deionized water. Exposure to PFPeS, PFHxS, PFHpS, or PFOS resulted in a shared toxicity phenotype characterized by body axis and swim bladder defects and hyperactivity. CONCLUSIONS: All emerging fluoroether PFAS tested were negative for evaluated outcomes. Two unique toxicity signatures were identified arising from structurally dissimilar PFAS. Among sulfonic acid aliphatic PFAS, chemical potencies were correlated with increasing carbon chain length for developmental neurotoxicity, but not developmental toxicity. This study identified relationships between chemical structures and in vivo phenotypes that may arise from shared mechanisms of PFAS toxicity. These data suggest that developmental neurotoxicity is an important end point to consider for this class of widely occurring environmental chemicals.

1,1,2,2-tetrafluoro-2-[1,1,1,2,3,3-hexafluoro-3-(1,1,2,2-tetrafluoroethoxy)propan-2-yl]oxyethane-1-sulfonic acid (PFESA-BP2) was first detected in 2012 in the Cape Fear River downstream of an industrial manufacturing facility. It was later detected in the finished drinking water of municipalities using the Cape Fear River for their water supply. No toxicology data exist for this contaminant despite known human exposure. To address this data gap, mice were dosed with PFESA-BP2 at 0, 0.04, 0.4, 3, and 6 mg/kg-day for 7 days by oral gavage. As an investigative study, the final dose groups evolved from an original dose of 3 mg/kg which produced liver enlargement and elevated liver enzymes. The dose range was extended to explore a no effect level. PFESA-BP2 was detected in the sera and liver of all treated mice. Treatment with PFESA-BP2 significantly increased the size of the liver for all mice at 3 and 6 mg/kg-day. At the 6 mg/kg-day dose, the liver more than doubled in size compared to the control group. Male mice treated with 3 and 6 mg/kg-day and females treated with 6 mg/kg-day demonstrated significantly elevated serum markers of liver injury including alanine aminotransferase (ALT), glutamate dehydrogenase (GLDH), and liver/body weight percent. The percent of PFESA-BP2 in serum relative to the amount administered was similar in male and female mice, ranged from 9 to 13 %, and was not related to dose. The percent accumulation in the liver of the mice varied by sex (higher in males), ranged from 30 to 65 %, and correlated positively with increasing dose level.

Oxidative stress (OS) contributes to the neurological and cardio/pulmonary effects caused by adverse metabolic states and air pollutants such as ozone (O3). This study explores the interactive effects of O3 and diet (high-fructose (FRUC) or high–fat (FAT)) on OS in different rat brain regions. In acute exposure, there was a decrease in markers of reactive oxygen species (ROS) production in some brain regions by diet and not by O3. Total antioxidant substances (TAS) were increased in the cerebellum (CER) and frontal cortex (FC) and decreased in the striatum (STR) by both diets irrespective of O3 exposure. Protein carbonyls (PC) and total aconitase decreased in some brain regions irrespective of exposure. Following subacute exposure, an increase in markers of ROS was observed in both diet groups. TAS was increased in the FC (FAT only) and there was a clear O3 effect where TAS was increased in the FC and STR. Diet increased PC formation within the CER in the FAT group, while the hippocampus showed a decrease in PC after O3 exposure in controls. In general, these results indicate that diet/O3 did not have a global effect on brain OS parameters, but showed some brain region- and OS parameter-specific effects by diets.

Cylindrospermopsin (CYN) is a toxin associated with numerous species of freshwater cyanobacteria throughout the world. It is postulated to have caused an episode of serious illnesses in Australia through treated drinking water, as well as lethal effects in livestock exposed to water from farm ponds. Toxicity included effects indicative of both hepatic and renal dysfunction. In humans, symptoms progressed from initial hepatomegaly, vomiting, and malaise to acidosis and hypokalemia, bloody diarrhea, and hyperemia in mucous membranes. Laboratory animal studies predominantly involved the intraperitoneal (i.p.) route of administration and confirmed this pattern of toxicity with changes in liver enzyme activities and histopathology consistent with hepatic injury and adverse renal effects. The aim of this study was designed to assess subchronic oral exposure (90 d) of purified CYN from 75 to 300 µg/kg/d in mouse. At the end of the dosing period, examinations of animals noted (1) elevated organ to body weight ratios of liver and kidney at all dose levels, (2) treatment-related increases in serum alanine aminotransferase (ALT) activity, (3) decreased blood urea nitrogen (BUN) and cholesterol concentrations in males, and (4) elevated monocyte counts in both genders. Histopathological alterations included hepatocellular hypertrophy and cord disruption in the liver, as well as renal cellular hypertrophy, tubule dilation, and cortical tubule lesions that were more prominent in males. A series of genes were differentially expressed including Bax (apoptosis), Rpl6 (tissue regeneration), Fabp4 (fatty acid metabolism), and Proc (blood coagulation). Males were more sensitive to many renal end points suggestive of toxicity. At the end of exposure, toxicity was noted at all dose levels, and the 75 µg/kg group exhibited significant effects in liver and kidney/body weight ratios, reduced BUN, increased serum monocytes, and multiple signs of histopathology indicating that a no-observed-adverse-effect level could not be determined for any dose level.

Emissions from oil fires associated with the “Deepwater Horizon” explosion and oil discharge that began on April 20, 2010 in the Gulf of Mexico were analyzed chemically to only a limited extent at the time but were shown to induce oxidative damage in vitro and in mice. To extend this work, we burned oil floating on sea water and performed extensive chemical analyses of the emissions (Gullett et al., Marine Pollut Bull, in press, 2017 ). Here, we examine the ability of a dichloromethane extract of the particulate material with an aerodynamic size ≤ 2.5 µm (PM 2.5 ) from those emissions to induce oxidative damage in human lung cells in vitro and mutagenicity in 6 strains of Salmonella. The extract had a percentage of extractable organic material (EOM) of 7.0% and increased expression of the heme oxygenase ( HMOX1 ) gene in BEAS-2B cells after exposure for 4 hr at 20 µg of EOM/ml. However, the extract did not alter mitochondrial respiration rate as measured by extracellular flux analysis. The extract was most mutagenic in TA100 +S9, indicative of a role for polycyclic aromatic hydrocarbons (PAHs), reflective of the high concentrations of PAHs in the emissions (1 g/kg of oil consumed). The extract had a mutagenicity emission factor of 1.8 ± 0.1 × 10 5 revertants/megajoule thermal in TA98 +S9, which was greater than that of diesel exhaust and within an order of magnitude of open burning of wood and plastic. Thus, organics from PM 2.5 of burning oil can induce oxidative responses in human airway epithelial cells and are highly mutagenic. Environ. Mol. Mutagen. 58:162–171, 2017. © 2017 Wiley Periodicals, Inc.

Epidemiological and experimental data suggest that obesity exacerbates the health effects of air pollutants such as ozone (O3). Maternal inactivity and calorically rich diets lead to offspring that show signs of obesity. Exacerbated O3 susceptibility of offspring could thus be manifested by maternal obesity. Thirty-day-old female Long-Evans rats were fed a control (CD) or high-fat (HF) (60% calories) diet for 6 wks and then bred. GD1 rats were then housed with a running wheel (RW) or without a wheel (SED) until parturition, creating four groups of offspring: CD-SED, CD-RW, HF-SED and HF-RW. HF diet was terminated at PND 35 and all offspring were placed on CD. Body weight and %fat of dams were greatest in order; HF-SED > HF-RW > CD-SED > CD-RW. Adult offspring were exposed to O3 for two consecutive days (0.8 ppm, 4 h/day). Glucose tolerance tests (GTT), ventilatory parameters (plethysmography), and bronchoalveolar fluid (BALF) cell counts and protein biomarkers were performed to assess response to O3. Exercise and diet altered body weight and %fat of young offspring. GTT, ventilation and BALF cell counts were exacerbated by O3 with responses markedly exacerbated in males. HF diet and O3 led to significant exacerbation of several BALF parameters: total cell count, neutrophils and lymphocytes were increased in male HF-SED versus CD-SED. Males were hyperglycemic after O3 exposure and exhibited exacerbated GTT responses. Ventilatory dysfunction was also exacerbated in males. Maternal exercise had minimal effects on O3 response. The results of this exploratory study suggest a link between maternal obesity and susceptibility to O3 in their adult offspring in a sex-specific manner.

Current strategies for predicting carcinogenic mode of action for nongenotoxic chemicals are based on identification of early key events in toxicity pathways. The goal of this study was to evaluate short-term key event indicators resulting from exposure to androstenedione (A4), an androgen receptor agonist and known liver carcinogen in mice. Liver cancer is more prevalent in men compared with women, but androgen-related pathways underlying this sex difference have not been clearly identified. Short-term hepatic effects of A4 were compared with reference agonists of the estrogen receptor (ethinyl estradiol, EE) and glucocorticoid receptor (prednisone, PRED). Male B6C3F1 mice were exposed for 7 or 28 days to A4, EE, or PRED. EE increased and PRED suppressed hepatocyte proliferation, while A4 had no detectable effects. In a microarray analysis, EE and PRED altered >3000 and >670 genes, respectively, in a dose-dependent manner, whereas A4 did not significantly alter any genes. Gene expression was subsequently examined in archival liver samples from male and female B6C3F1 mice exposed to A4 for 90 days. A4 altered more genes in females than males and did not alter expression of genes linked to activation of the mitogenic xenobiotic receptors AhR, CAR, and PPARα in either sex. A gene expression biomarker was used to show that in female mice, the high dose of A4 activated the growth hormone-regulated transcription factor STAT5b, which controls sexually dimorphic gene expression in the liver. These findings suggest that A4 induces subtle age-related effects on STAT5b signaling that may contribute to the higher risk of liver cancer in males compared with females.

Emissions from oil fires associated with the “Deepwater Horizon” explosion and oil discharge that began on April 20, 2010 in the Gulf of Mexico were analyzed chemically to only a limited extent at the time but were shown to induce oxidative damage in vitro and in mice. To extend this work, we burned oil floating on sea water and performed extensive chemical analyses of the emissions (Gullett et al., Marine Pollut Bull, in press, ). Here, we examine the ability of a dichloromethane extract of the particulate material with an aerodynamic size ≤ 2.5 µm (PM2.5) from those emissions to induce oxidative damage in human lung cells in vitro and mutagenicity in 6 strains of Salmonella. The extract had a percentage of extractable organic material (EOM) of 7.0% and increased expression of the heme oxygenase (HMOX1) gene in BEAS‐2B cells after exposure for 4 hr at 20 µg of EOM/ml. However, the extract did not alter mitochondrial respiration rate as measured by extracellular flux analysis. The extract was most mutagenic in TA100 +S9, indicative of a role for polycyclic aromatic hydrocarbons (PAHs), reflective of the high concentrations of PAHs in the emissions (1 g/kg of oil consumed). The extract had a mutagenicity emission factor of 1.8 ± 0.1 × 105 revertants/megajoulethermal in TA98 +S9, which was greater than that of diesel exhaust and within an order of magnitude of open burning of wood and plastic. Thus, organics from PM2.5 of burning oil can induce oxidative responses in human airway epithelial cells and are highly mutagenic. Environ. Mol. Mutagen. 58:162–171, 2017. © 2017 Wiley Periodicals, Inc.

The compound BMAA (β-N-methylamino-L-alanine) has been postulated to play a significant role in four serious neurological human diseases: Amyotrophic Lateral Sclerosis/Parkinsonism Dementia Complex (ALS/PDC) found on Guam, and ALS, Parkinsonism, and dementia that occur globally. ALS/PDC with symptoms of all three diseases first came to the attention of the scientific community during and after World War II. It was initially associated with cycad flour used for food because BMAA is a product of symbiotic cycad root-dwelling cyanobacteria. Human consumption of flying foxes that fed on cycad seeds was later suggested as a source of BMAA on Guam and a cause of ALS/PDC. Subsequently, the hypothesis was expanded to include a causative role for BMAA in other neurodegenerative diseases including Alzheimer's disease (AD) through exposures attributed to proximity to freshwaters and/or consumption of seafood due to its purported production by most species of cyanobacteria. The hypothesis that BMAA is the critical factor in the genesis of these neurodegenerative diseases received considerable attention in the medical, scientific, and public arenas. This review examines the history of ALS/PDC and the BMAA-human disease hypotheses; similarities and differences between ALS/PDC and the other diseases with similar symptomologies; the relationship of ALS/PDC to other similar diseases, studies of BMAA-mediated effects in lab animals, inconsistencies and data gaps in the hypothesis; and other compounds and agents that were suggested as the cause of ALS/PDC on Guam. The review concludes that the hypothesis of a causal BMAA neurodegenerative disease relationship is not supported by existing data.

Pools and spas are enjoyed throughout the world for exercise and relaxation. However, there are no previous studies on mutagenicity of disinfected spa (hot tub) waters or comprehensive identification of disinfection byproducts (DBPs) formed in spas. Using 28 water samples from seven sites, we report the first integrated mutagenicity and comprehensive analytical chemistry of spas treated with chlorine, bromine, or ozone, along with pools treated with these same disinfectants. Gas chromatography (GC) with high-resolution mass spectrometry, membrane-introduction mass spectrometry, and GC-electron capture detection were used to comprehensively identify and quantify DBPs and other contaminants. Mutagenicity was assessed by the Salmonella mutagenicity assay. More than 100 DBPs were identified, including a new class of DBPs, bromoimidazoles. Organic extracts of brominated pool/spa waters were 1.8× more mutagenic than chlorinated ones; spa waters were 1.7× more mutagenic than pools. Pool and spa samples were 2.4 and 4.1× more mutagenic, respectively, than corresponding tap waters. The concentration of the sum of 21 DBPs measured quantitatively increased from finished to tap to pool to spa; and mutagenic potency increased from finished/tap to pools to spas. Mutagenic potencies of samples from a chlorinated site correlated best with brominated haloacetic acid concentrations (Br-HAAs) (r = 0.98) and nitrogen-containing DBPs (N-DBPs) (r = 0.97) and the least with Br-trihalomethanes (r = 0.29) and Br-N-DBPs (r = 0.04). The mutagenic potencies of samples from a brominated site correlated best (r = 0.82) with the concentrations of the nine HAAs, Br-HAAs, and Br-DBPs. Human use increased significantly the DBP concentrations and mutagenic potencies for most pools and spas. These data provide evidence that human precursors can increase mutagenic potencies of pools and spas and that this increase is associated with increased DBP concentrations.

Emissions from solid-fuel burning cookstoves are associated with 3 to 4 million premature deaths annually and contribute significantly to impacts on climate. Pellet-fueled gasifier stoves have some emission factors (EFs) approaching those of gas-fuel (liquid petroleum gas) stoves; however, their emissions have not been evaluated for biological effects. Here we used a new International Organization for Standardization (ISO) testing protocol to determine pollutant- and mutagenicity-EFs for a stove designed for pellet fuel, the Mimi Moto, and for two other forced-draft stoves, Xunda and Philips HD4012, burning pellets of hardwood or peanut hulls. The Salmonella assay-based mutagenicity-EFs (revertants/megajouledelivered) spanned three orders of magnitude and correlated highly (r = 0.99; n = 5) with EFs of the sum of 32 particle-phase polycyclic aromatic hydrocarbons (PAHs). The Mimi Moto/hardwood pellet combination had total-PAH- and mutagenicity-EFs 99.2 and 96.6% lower, respectively, compared to data published previously for the Philips stove burning non-pelletized hardwood, and 100 and 99.8% lower, respectively, compared to those of a wood-fueled three-stone fire. The Xunda burning peanut hull pellets had the highest fuel energy-based mutagenicity-EF (revertants/megajoulethermal) of the pellet stove/fuel combinations tested, which was between that of diesel exhaust, a known human carcinogen, and a natural-draft wood stove. Although the Mimi Moto burning hardwood pellets had the lowest fuel energy-based mutagenicity-EF, this value was between that of utility coal and utility wood boilers. This advanced stove/fuel combination has the potential to greatly reduce emissions in contrast to a traditional stove, but adequate ventilation is required to approach acceptable levels of indoor air quality.

Current strategies for predicting adverse health outcomes of environmental chemicals are centered on early key events in toxicity pathways. However, quantitative relationships between early molecular changes in a given pathway and later health effects are often poorly defined. The goal of this study was to evaluate short-term key event indicators using qualitative and quantitative methods in an established pathway of mouse liver tumorigenesis mediated by peroxisome proliferator-activated receptor alpha (PPARα). Male B6C3F1 mice were exposed for 7 days to di (2-ethylhexyl) phthalate (DEHP), di-n-octyl phthalate (DNOP), and n-butyl benzyl phthalate (BBP), which vary in PPARα activity and liver tumorigenicity. Each phthalate increased expression of select PPARα target genes at 7 days, while only DEHP significantly increased liver cell proliferation labeling index (LI). Transcriptional benchmark dose (BMDT) estimates for dose-related genomic markers stratified phthalates according to hypothetical tumorigenic potencies, unlike BMDs for non-genomic endpoints (relative liver weights or proliferation). The 7-day BMDT values for Acot1 as a surrogate measure for PPARα activation were 29, 370, and 676 mg/kg/day for DEHP, DNOP, and BBP, respectively, distinguishing DEHP (liver tumor BMD of 35 mg/kg/day) from non-tumorigenic DNOP and BBP. Effect thresholds were generated using linear regression of DEHP effects at 7 days and 2-year tumor incidence values to anchor early response molecular indicators and a later phenotypic outcome. Thresholds varied widely by marker, from 2-fold (Pdk4 and proliferation LI) to 30-fold (Acot1) induction to reach hypothetical tumorigenic expression levels. These findings highlight key issues in defining thresholds for biological adversity based on molecular changes.