Metabolism of 3-Chlorobiphenyl (PCB 2) in a Human-Relevant Cell Line: Evidence of Dechlorinated Metabolites.

Lower chlorinated polychlorinated biphenyls (LC-PCBs) and their metabolites make up a class of environmental pollutants implicated in a range of adverse outcomes in humans; however, the metabolism of LC-PCBs in human models has received little attention. Here we characterize the metabolism of PCB 2 (3-chlorobiphenyl), an environmentally relevant LC-PCB congener, in HepG2 cells with in silico prediction and nontarget high-resolution mass spectrometry. Twenty PCB 2 metabolites belonging to 13 metabolite classes, including five dechlorinated metabolite classes, were identified in the cell culture media from HepG2 cells exposed for 24 h to 10 µM or 3.6 nM PCB 2. The PCB 2 metabolite profiles differed from the monochlorinated metabolite profiles identified in samples from an earlier study with PCB 11 (3,3'-dichlorobiphenyl) under identical experimental conditions. A dechlorinated dihydroxylated metabolite was also detected in human liver microsomal incubations with monohydroxylated PCB 2 metabolites but not PCB 2. These findings demonstrate that the metabolism of LC-PCBs in human-relevant models involves the formation of dechlorination products. In addition, untargeted metabolomic analyses revealed an altered bile acid biosynthesis in HepG2 cells. Our results indicate the need to study the disposition and toxicity of complex PCB 2 metabolites, including novel dechlorinated metabolites, in human-relevant models.

The Aryl hydrocarbon receptor mediates reproductive toxicity of polychlorinated biphenyl congener 126 in rats.

Polychlorinated biphenyls (PCBs) are endocrine disrupting chemicals with documented, though mechanistically ill-defined, reproductive toxicity. The toxicity of dioxin-like PCBs, such as PCB126, is mediated via the aryl hydrocarbon receptor (AHR) in non-ovarian tissues. The goal of this study was to examine the uterine and ovarian effects of PCB126 and test the hypothesis that the AHR is required for PCB126-induced reproductive toxicity. Female Holzman-Sprague Dawley wild type (n = 14; WT) and Ahr knock out (n = 11; AHR-/-) rats received a single intraperitoneal injection of either corn oil vehicle (5 ml/kg: WT_O and AHR-/-_O) or PCB126 (1.63 mg/kg in corn oil: WT_PCB and AHR-/-_PCB) at four weeks of age. The estrous cycle was synchronized and ovary and uterus were collected 28 days after exposure. In WT rats, PCB126 exposure reduced (P < 0.05) body and ovary weight, uterine gland number, uterine area, progesterone, 17ß-estradiol and anti-Müllerian hormone level, secondary and antral follicle and corpora lutea number but follicle stimulating hormone level increased (P < 0.05). In AHR-/- rats, PCB126 exposure increased (P ≤ 0.05) circulating luteinizing hormone level. Ovarian or uterine mRNA abundance of biotransformation, and inflammation genes were altered (P < 0.05) in WT rats due to PCB126 exposure. In AHR-/- rats, the transcriptional effects of PCB126 were restricted to reductions (P < 0.05) in three inflammatory genes. These findings support a functional role for AHR in the female reproductive tract, illustrate AHR's requirement in PCB126-induced reprotoxicity, and highlight the potential risk of dioxin-like compounds on female reproduction.

Characterization of the Metabolic Pathways of 4-Chlorobiphenyl (PCB3) in HepG2 Cells Using the Metabolite Profiles of Its Hydroxylated Metabolites.

The characterization of the metabolism of lower chlorinated PCB, such as 4-chlorobiphenyl (PCB3), is challenging because of the complex metabolite mixtures formed in vitro and in vivo. We performed parallel metabolism studies with PCB3 and its hydroxylated metabolites to characterize the metabolism of PCB3 in HepG2 cells using nontarget high-resolution mass spectrometry (Nt-HRMS). Briefly, HepG2 cells were exposed for 24 h to 10 µM PCB3 or its seven hydroxylated metabolites in DMSO or DMSO alone. Six classes of metabolites were identified with Nt-HRMS in the culture medium exposed to PCB3, including monosubstituted metabolites at the 3'-, 4'-, 3-, and 4- (1,2-shift product) positions and disubstituted metabolites at the 3',4'-position. 3',4'-Di-OH-3 (4'-chloro-3,4-dihydroxybiphenyl), which can be oxidized to a reactive and toxic PCB3 quinone, was a central metabolite that was rapidly methylated. The resulting hydroxylated-methoxylated metabolites underwent further sulfation and, to a lesser extent, glucuronidation. Metabolomic analyses revealed an altered tryptophan metabolism in HepG2 cells following PCB3 exposure. Some PCB3 metabolites were associated with alterations of endogenous metabolic pathways, including amino acid metabolism, vitamin A (retinol) metabolism, and bile acid biosynthesis. In-depth studies are needed to investigate the toxicities of PCB3 metabolites, especially the 3',4'-di-OH-3 derivatives identified in this study.

Atropselective Partitioning of Polychlorinated Biphenyls in a HepG2 Cell Culture System: Experimental and Modeling Results.

Cell culture models are used to study the toxicity of polychlorinated biphenyls (PCBs); however, it is typically unknown how much PCB enters the cells and, for chiral PCBs, if the partitioning is atropselective. We investigated the partitioning of racemic PCB 91, PCB 95, PCB 132, and PCB 136 in HepG2 cells following a 72 h incubation. PCBs were present in the cell culture medium (60.7-88.8%), cells (8.0-14.6%), and dishes (2.3-7.8%) and displayed atropisomeric enrichment in the cells (enantiomeric fraction [EF] = 0.55-0.77) and dishes (EF = 0.53-0.68). Polyparameter linear free energy relationships coupled with a composition-based model provided a good estimate of the PCB levels in the cells and cell culture medium. The free concentration was subsequently used to extrapolate from the nominal cell culture concentration to PCB tissue levels and vice versa. This approach can be used for in vitro-in vivo extrapolations for all 209 PCB congeners. However, this model (and modified models based on descriptors incorporating atropselective interactions, i.e., relative retention times on chiral columns) did not predict the atropselective partitioning in the cell culture system. Improved chemical descriptors that account for the atropselective binding of PCBs to biological macromolecules are, therefore, needed to predict the atropselective partitioning of PCBs in biological systems.

3,3'-Dichlorobiphenyl Is Metabolized to a Complex Mixture of Oxidative Metabolites, Including Novel Methoxylated Metabolites, by HepG2 Cells.

3,3'-Dichlorobiphenyl (PCB 11) is a byproduct of industrial processes and detected in environmental samples. PCB 11 and its metabolites are present in human serum, and emerging evidence demonstrates that PCB 11 is a developmental neurotoxicant. However, little is known about the metabolism of PCB 11 in humans. Here, we investigated the metabolism of PCB 11 and the associated metabolomics changes in HepG2 cells using untargeted high-resolution mass spectrometry. HepG2 cells were exposed for 24 h to PCB 11 in DMSO or DMSO alone. Cell culture media were analyzed with ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry. Thirty different metabolites were formed by HepG2 cells exposed to 10 µM PCB 11, including monohydroxylated, dihydroxylated, methoxylated-hydroxylated, and methoxylated-dihydroxylated metabolites and the corresponding sulfo and glucuronide conjugates. The methoxylated PCB metabolites were observed for the first time in a human-relevant model. 4-OH-PCB 11 (3,3'-dichlorobiphenyl-4-ol) and the corresponding catechol metabolite, 4,5-di-OH-PCB 11 (3',5-dichloro-3,4-dihydroxybiphenyl), were unambiguously identified based on liquid and gas chromatographic analyses. PCB 11 also altered several metabolic pathways, in particular vitamin B6 metabolism. These results demonstrate that complex PCB 11 metabolite profiles are formed in HepG2 cells that warrant further toxicological investigation, particularly since catechol metabolites are likely reactive and toxic.

Comprehensive Subchronic Inhalation Toxicity Assessment of an Indoor School Air Mixture of PCBs.

Few in vivo inhalation studies have explored the toxicity of environmentally relevant mixtures of polychlorinated biphenyls (PCBs). The manufacture of industrial PCBs was banned in 1978, but PCBs continue to be formed in industrial and consumer products. Schools represent a significant source of airborne exposures to legacy and nonlegacy PCBs, placing children at risk. To evaluate the impact of these exposures, we generated an airborne mixture of PCBs, called the School Air Mixture (SAM), to match the profile of an older school from our adolescent cohort study. Female Sprague-Dawley rats were exposed either to SAM or filtered air in nose-only exposure systems, 4 h/day for 4 weeks. Congener-specific air and tissue PCB profiles were assessed using gas chromatography with tandem mass spectrometry (GC-MS/MS). PCB exposures recapitulated the target school air profile with a similarity coefficient, cos θ of 0.83. PCB inhalation yielded µg/g ∑209 PCB levels in tissues. Neurobehavioral testing demonstrated a modest effect on spatial learning and memory in SAM-exposed rats. PCB exposure induced oxidative stress in the liver and lungs, affected the maturational stages of hematopoietic stem cells, reduced telomerase activity in bone marrow cells, and altered the gut microbiota. This is the first study to emulate PCB exposures in a school and comprehensively evaluate toxicity.

Low-level arsenic exposure from drinking water is associated with prostate cancer in Iowa.

Inorganic arsenic is a toxic naturally occurring element in soil and water in many regions of the US including the Midwest. Prostate cancer is the second most common type of cancer in men in Iowa, surpassed only by non-melanotic skin cancer. Epidemiology studies have evaluated arsenic exposure from drinking water and prostate cancer, but most have focused on high-level exposures outside the US. As drinking water from groundwater sources is a major source of arsenic exposure, we conducted an ecologic study to evaluate prostate cancer and arsenic in drinking water from public water sources and private wells in Iowa, where exposure levels are low, but duration of exposure can be long. Arsenic data from public water systems were obtained from the Iowa Safe Drinking Water Information System for the years 1994-2003 and for private wells from two Iowa Well Water Studies, the Iowa Community Private Well Study (ICPWS, 2002-2003) and Iowa Statewide Rural Well Water Survey Phase 2 (SWIRL2, 2006-2008) that provided data for 87 Iowa counties. Prostate cancer incidence data from 2009 to 2013 for Iowa were obtained from Surveillance, Epidemiology and End Results' SEER*Stat software. County averages of water arsenic levels varied from 1.08 to 18.6 ppb, with three counties above the current 10 ppb limit. Based on the tertiles of arsenic levels, counties were divided into three groups: low (1.08-2.06 ppb), medium (2.07-2.98 ppb), and high (2.99-18.6 ppb). Spatial Poisson regression modeling was conducted to estimate the risk ratios (RR) of prostate cancer by tertiles of arsenic level at a county level, adjusted for demographic and risk factors. The RR of prostate cancer were 1.23 (95% CI, 1.16-1.30) and 1.28 (95% CI, 1.21-1.35) in the medium and high groups, respectively, compared to the low group after adjusting for risk factors. The RR increased to 1.36 (95% CI, 1.28-1.45) in the high group when analyses were restricted to aggressive prostate cancers (Gleason score ≥ 7). This study shows a significant dose-dependent association between low-level arsenic exposure and prostate cancer, and if this result is replicated in future individual-level studies, may suggest that 10 ppb is not protective for human health.

Metabolism and metabolites of polychlorinated biphenyls.

Abstract The metabolism of polychlorinated biphenyls (PCBs) is complex and has an impact on toxicity, and thereby on the assessment of PCB risks. A large number of reactive and stable metabolites are formed in the processes of biotransformation in biota in general, and in humans in particular. The aim of this document is to provide an overview of PCB metabolism, and to identify the metabolites of concern and their occurrence. Emphasis is given to mammalian metabolism of PCBs and their hydroxyl, methylsulfonyl, and sulfated metabolites, especially those that persist in human blood. Potential intracellular targets and health risks are also discussed.

Does dietary copper supplementation enhance or diminish PCB126 toxicity in the rodent liver?

Copper is essential for the function of the mitochondrial electron transport chain and several antioxidant proteins. However, in its free form copper can participate in Fenton-like reactions that produce reactive hydroxyl radicals. Aryl-hydrocarbon receptor (AhR) agonists, including the most potent polychlorinated biphenyl (PCB) congener, 3,3',4,4',5-pentachlorobiphenyl (PCB126), increase copper levels in rodent livers. This is accompanied by biochemical and toxic changes. To assess the involvement of copper in PCB toxicity, male Sprague-Dawley rats were fed an AIN-93G diet with differing dietary copper levels: low (2 ppm), adequate (6 ppm), and high (10 ppm). After three weeks, rats from each group were given a single ip injection of corn oil (control), 1, or 5 µmol/kg body weight PCB126. Two weeks following injections, biochemical and morphological markers of hepatic toxicity, trace metal status, and hepatic gene expression of metalloproteins were evaluated. Increasing dietary copper was associated with elevated tissue levels of copper and ceruloplasmin. In the livers of PCB126-treated rats, the hallmark signs of AhR activation were present, including increased cytochrome P450 and lipid levels and decreased glutathione. In addition, a doubling of hepatic copper levels was seen, and overall metal homeostasis was disturbed, resulting in decreased hepatic selenium, manganese, zinc, and iron. Expression of key metalloproteins was either decreased (cytochrome c oxidase), unchanged (ceruloplasmin and CuZnSOD), or increased (tyrosinase and metallothioneins 1 and 2) with exposure to PCB126. Increases in metallothionein may contribute/reflect the increased copper seen. Alterations in dietary copper did not amplify or abrogate the hepatic toxicity of PCB126. PCB126 toxicity, i.e., oxidative stress and steatosis, is clearly associated with disturbed metal homeostasis. Understanding the mechanisms of this disturbance may provide tools to prevent liver toxicity by other AhR agonists.

A Case for Accelerating Standards to Achieve the FAIR Principles of Environmental Health Research Experimental Data.

BACKGROUND: Funding agencies, publishers, and other stakeholders are pushing environmental health science investigators to improve data sharing; to promote the findable, accessible, interoperable, and reusable (FAIR) principles; and to increase the rigor and reproducibility of the data collected. Accomplishing these goals will require significant cultural shifts surrounding data management and strategies to develop robust and reliable resources that bridge the technical challenges and gaps in expertise. OBJECTIVE: In this commentary, we examine the current state of managing data and metadata-referred to collectively as (meta)data-in the experimental environmental health sciences. We introduce new tools and resources based on in vivo experiments to serve as examples for the broader field. METHODS: We discuss previous and ongoing efforts to improve (meta)data collection and curation. These include global efforts by the Functional Genomics Data Society to develop metadata collection tools such as the Investigation, Study, Assay (ISA) framework, and the Center for Expanded Data Annotation and Retrieval. We also conduct a case study of in vivo data deposited in the Gene Expression Omnibus that demonstrates the current state of in vivo environmental health data and highlights the value of using the tools we propose to support data deposition. DISCUSSION: The environmental health science community has played a key role in efforts to achieve the goals of the FAIR guiding principles and is well positioned to advance them further. We present a proposed framework to further promote these objectives and minimize the obstacles between data producers and data scientists to maximize the return on research investments. https://doi.org/10.1289/EHP11484.

Characterization of Arsenic and Atrazine Contaminations in Drinking Water in Iowa: A Public Health Concern.

Arsenic and atrazine are two water contaminants of high public health concern in Iowa. The occurrence of arsenic and atrazine in drinking water from Iowa's private wells and public water systems was investigated over several decades. In this study, the percentages of detection and violation of regulations were compared over region, season, and water source, and factors affecting the detection and concentration of arsenic and atrazine were analyzed using a mixed-effects model. Atrazine contamination in drinking water was found to vary by region, depending on agricultural usage patterns and hydrogeological features. The annual median atrazine levels of all public water systems were below the drinking water standard of 3 ppb in 2001-2014. Around 40% of public water systems contained arsenic at levels > 1 ppb in 2014, with 13.8% containing arsenic at levels of 5-10 ppb and 2.6% exceeding 10 ppb. This unexpected result highlights the ongoing public health threat posed by arsenic in drinking water in Iowa, emphasizing the need for continued monitoring and mitigation efforts to reduce exposure and associated health risks. Additionally, an atrazine metabolite, desethylatrazine, should be monitored to obtain a complete account of atrazine exposure and possible health effects.

PCB126 induced toxic actions on liver energy metabolism is mediated by AhR in rats.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor involved in the regulation of biological responses to more planar aromatic hydrocarbons, like TCDD. We previously described the sequence of events following exposure of male rats to a dioxin-like polychlorinated biphenyl (PCB) congener, 3,3',4,4',5-pentachlorobiphenyl (PCB126), that binds avidly to the AhR and causes various types of toxicity including metabolic syndrome, fatty liver, and disruption of energy homeostasis. The purpose of this study was, to investigate the role of AhR to mediate those toxic manifestations following sub-acute exposure to PCB126 and to examine possible sex differences in effects. For this goal, we created an AhR knockout (AhR-KO) model using CRISPR/Cas9. Comparison was made to the wild type (WT) male and female Holtzman Sprague Dawley rats. Rats were injected with a single IP dose of corn oil vehicle or 5 µmol/kg PCB126 in corn oil and necropsied after 28 days. PCB126 caused significant weight loss, reduced relative thymus weights, and increased relative liver weights in WT male and female rats, but not in AhR-KO rats. Similarly, significant pathologic changes were visible which included necrosis and regeneration in female rats, micro- and macro-vesicular hepatocellular vacuolation in males, and a paucity of glycogen in livers of both sexes in WT rats only. Hypoglycemia and lower IGF1, and reduced serum non-esterified fatty acids (NEFAs) were found in serum of both sexes of WT rats, low serum cholesterol levels only in the females, and no changes in AhR-KO rats. The expression of genes encoding enzymes related to xenobiotic metabolism (e.g. CYP1A1), gluconeogenesis, glycogenolysis, and fatty acid oxidation were unaffected in the AhR-KO rats following PCB126 exposure as opposed to WT rats where expression was significantly upregulated (PPARα, females only) or downregulated suggesting a disrupted energy homeostasis. Interestingly, Acox2, Hmgcs, G6Pase and Pc were affected in both sexes, the gluconeogenesis and glucose transporter genes Pck1, Glut2, Sds, and Crem only in male WT-PCB rats. These results show the essential role of the AhR in glycogenolysis, gluconeogenesis, and fatty acid oxidation, i.e. in the regulation of energy production and homeostasis, but also demonstrate a significant difference in the effects of PCB126 in males verses females, suggesting higher vulnerability of glucose homeostasis in males and more changes in fatty acid/lipid homeostasis in females. These differences in effects, which may apply to more/all AhR agonists, should be further analyzed to identify health risks to specific groups of highly exposed human populations.

Species difference in the regulation of cytochrome P450 2S1: lack of induction in rats by the aryl hydrocarbon receptor agonist PCB126.

CYP2S1 is an evolutionarily conserved, mainly extra-hepatic member of the CYP2 family and proposed to be regulated by the aryl hydrocarbon receptor (AhR). The present study explores AhR's regulation of CYP2S1 in male Sprague Dawley rats using PCB126 (3,3',4,4',5-pentachlorobiphenyl), the most potent AhR agonist among the PCBs. Additionally, CYP2S1 expression was examined after treatments with the classic CYP-inducers ß-naphthoflavone (ß-NF, AhR activator), phenobarbital (PB, CAR activator) and dexamethasone (Dex, PXR activator). CYP2S1 and CYP1A1/2, CYP1B1, CYP2B and CYP3A mRNAs were measured in liver, lung, spleen, stomach, kidney, and thymus at different time points. Constitutive CYP2S1 was expressed at comparable levels to other CYPs with the highest expression levels in stomach, kidney and lung. CYP2S1 mRNA was only non-significantly elevated by ß-NF in liver tissues. PCB126 did not increase CYP2S1 mRNA in any organ and at any time point examined despite a significant induction of CYP1 genes. PCB126 reduced CYP2S1 mRNA by 40% (not significant) from the 7th post-exposure day in thymus. PB and Dex had no effect on CYP2S1 mRNA levels. These observations show that in this model CYP2S1 is not, or only weakly, regulated by AhR and not induced by CAR or PXR activators.

Polychlorinated Biphenyl (PCB) carcinogenicity with special emphasis on airborne PCBs.

Polychlorinated biphenyls (PCBs) are industrial chemicals used in various applications requiring chemical stabilityand have now become widely dispersed. Their characteristics of persistence, low water/higher lipid solubility, contribute to their ability to bioconcentrate and bioaccumulate. Traditionally PCBs have been regulated as food contaminants and the general population is primarily exposed by that route. PCBs in foodstuffs are generally higher chlorinated, resistant to metabolic breakdown, and elicit toxic changes that are thought to be predominantly receptor/parent PCB-driven. But for certain occupational exposures, and for those persons residing or working in contaminated buildings, and in large cities, an inhalation route of exposure may predominate. Airborne PCBs are, in contrast to foodborne PCBs, lower chlorinated, more volatile, and subject to metabolic attack. In this review, we have explored (geno-) toxic manifestations of PCBs typical of those found in air. Here metabolic conversion of the parent PCB to hydroxylated and other metabolic progeny appear to play a dominant role, especially in genotoxicity. We should be cognizant of the impact of exposures to airborne PCBs for those individuals who are occupationally exposed, for persons living near contaminated sites, for those who work or go to school in contaminated buildings, and especially cognizant of the young, the socio-economically disadvantaged and medically-underserved or nutritionally-deficient populations.

Association between Body Iron Status and Leukocyte Telomere Length, a Biomarker of Biological Aging, in a Nationally Representative Sample of US Adults.

BACKGROUND: Excess iron levels can induce oxidative stress and could therefore affect telomere attrition. However, little is known about the impact of body iron status on telomere length. OBJECTIVE: Our aim was to examine the association between serum ferritin concentrations, an indicator of body iron status, and leukocyte telomere length in US adults. DESIGN: We conducted a nationwide, population-based, cross-sectional study. PARTICIPANTS/SETTING: We used data from the National Health and Nutrition Examination Survey (NHANES) 1999-2002. We included 7,336 adults aged 20 years or older who had available data on serum ferritin levels and telomere length. High ferritin levels were defined as a serum ferritin level >200 ng/mL (449.4 pmol/L) in women and >300 ng/mL (674.1 pmol/L) in men. Low ferritin levels were defined as a serum ferritin level <30 ng/mL (67.4 pmol/L). MAIN OUTCOME MEASURES: Leukocyte telomere length was assayed using the quantitative polymerase chain reaction method. STATISTICAL ANALYSES: Linear regression with survey weights was performed to estimate the association between serum ferritin levels and telomere length. RESULTS: The prevalence of adults with high and low serum ferritin levels was 10.9% and 17.6%, respectively. High ferritin levels were inversely associated with telomere length compared to normal ferritin levels. After adjustment for demographic, socioeconomic and lifestyle factors, body mass index, C-reactive protein, and leukocyte cell type composition, the ß coefficient for log-transformed telomere length was -0.020 (standard error [SE]=0.009; P=0.047). The association was stronger in adults aged 65 years or older (ß coefficient -0.081, SE=0.017; P<0.001) than in adults 20 to 44 years old (ß coefficient -0.023, SE=0.019; P=0.24) or adults aged 45 to 64 years old (ß coefficient 0.024, SE=0.015; P=0.10) (P for interaction 0.003). Low ferritin levels were not significantly associated with telomere length compared with normal ferritin levels. CONCLUSIONS: In a US nationally representative population, high body iron status was associated with shorter telomeres, especially in adults aged 65 years or older.

Xenobiotic geometry and media pH determine cytotoxicity through solubility.

Polychlorinated biphenyls (PCBs), a class of 209 individual congeners, have become persistent and ubiquitous environmental contaminants. The health impacts of PCBs, such as cancer, cardiovascular disease, developmental toxicity, and neurotoxicity, have been widely reported, but for many of these, the mechanisms of toxicity are still poorly understood. Many mechanistic studies involve cultured cells where the biological activity is dependent upon the solubility of the xenobiotic. In the present study, we investigated the factors that determine solubility as measured by diffraction spectroscopy and have modeled, with semiempirical and ab initio molecular orbital methods, the dihedral angle and calculated the dipole moment of a series of monofluorinated analogues (F-PCBs 3) of 4-chlorobiphenyl (PCB 3) as model compounds in vacuum and in water. We found a strong positive correlation between the dihedral angle, the rotation energy, the cavitation energy, the solubility, and the cytotoxicity in three human cell lines. The dipole moment was of minor influence. We also determined the influence of pH changes in a medium containing 10% fetal bovine serum (FBS), changes that could be expected when cells in culture are removed from a CO 2 incubator even for a short time. We found that the solubility is strongly affected by the pH and that this effect is not reversed by subsequent pH readjustment. In a study examining cytotoxicity, we showed that the actual pH and the pH history of a medium containing FBS were of major influence. We suggest that pH-driven changes in the tertiary and quaternary structure of albumin are responsible. These observations have implications for studies of the biological activity of semisoluble compounds, like PCBs and related compounds.

Synthesis, physicochemical properties and in vitro cytotoxicity of nicotinic acid ester prodrugs intended for pulmonary delivery using perfluorooctyl bromide as vehicle.

This study explores perfluorooctyl bromide (PFOB) as a potential vehicle for the pulmonary delivery of a series of prodrugs of nicotinic acid using cell culture studies. The prodrugs investigated have PFOB-water (logK(p)=0.78 to >2.2), perfluoromethylcyclohexane-toluene (logK(p)=-2.62 to 0.13) and octanol-water (logK(p)=0.90-10.2) partition coefficients spanning several orders of magnitude. In confluent NCI-H358 human lung cancer cells, the toxicity of prodrugs administered in culture medium or PFOB depends on the medium of administration, with EC20's above 8 mM and 2.5 mM for culture medium and PFOB, respectively. Short-chain nicotinates administered both in PFOB and medium increase cellular NAD/NADP levels at 1mM nicotinate concentrations. Long-chain nicotinates, which could not be administered in medium due to their poor aqueous solubility, increased NAD/NADP levels if administered in PFOB at concentrations > or =10 mM. These findings suggest that even highly lipophilic prodrugs can partition out of the PFOB phase into cells, where nicotinic acid is released and converted to NAD. Thus, PFOB may be a novel and biocompatible vehicle for the delivery of lipophilic prodrugs of nicotinic acid and other drugs directly to the lung of laboratory animals and humans.

Effects of estradiol, PCB3, and their hydroxylated metabolites on proliferation, cell cycle, and apoptosis of human breast cancer cells.

Certain estradiol metabolites and industrial pollutants, like polychlorinated biphenyls, may play a more important role in enhancing breast cancer risk than 17ß-estradiol. The aim of this study was to compare the effects of 17ß-estradiol (E2) with that of the air pollutant 4-chlorobiphenyl (PCB3) and four of their hydroxylated metabolites on cell cycle, proliferation, and apoptosis in MCF-7 human breast cancer cells at concentrations of 0.1-10nM (E2, 2-OH-E2, and 4-OH-E2) and 0.3-300nM (PCB3, 4-OH-PCB3, and 3, 4-diOH-PCB3) and 24-260h of exposure. E2 increased cell proliferation and cells in S-phase at all time points. 2-OH-E2 and 4-OH-E2 had no effect on the cell cycle, but a stimulatory action on cell proliferation from 72 to 260h of exposure to 4-OH-E2 and at 260h to 2-OH-E2 was seen. E2 and its metabolites had no effect on apoptosis. PCB3 and 4-OH-PCB3 showed no effect on proliferation, apoptosis or cell cycle distribution at any concentration and time point. Longer time exposures to 3,4-di-OH-PCB at 300nM caused a decrease of cells and an increase in G2/M and apoptotic cells. These results confirm the proliferative effect of E2 and its metabolite 4-OH-E2 in estrogen receptor positive breast cancer cells, but show no mitogenic activity for PCB3 and 4-OH-PCB3. However, the cell cycle and apoptosis effects of 3,4-diOH-PCB3 need further analysis.

Metabolic Activation of PCBs to Carcinogens in Vivo - A Review.

Many higher-chlorinated biphenyls, persistent and predominant in foods, are active as promoters in hepatocarcinogenesis. Lower-chlorinated biphenyls, predominating in indoor and outdoor air, are more readily metabolized and therefore shorter lived, 'episodic' contaminants. Thus inhalation of such lower chlorinated biphenyls may expose humans to reactive, possibly genotoxic/carcinogenic intermediates. Lower chlorinated biphenyls may be metabolized via arene-oxides to mono- and dihydroxylated intermediates and further to (semi)quinones, highly reactive intermediates. Covalently bound lower chlorinated biphenyls have been detected in rodent tissues in vivo. We recently showed using the modified Solt-Farber foci assay that several mono- to tetrachlorinated biphenyls have initiating activity in the livers of rats. In a follow-up study of PCB3 (4-chlorobiphenyl) metabolites only one monohydroxy- and one quinoid- metabolite showed initiating activity, indicating that the metabolic activation of PCB3 proceeds via hydroxylation and oxidation to the 3,4-quinone, the ultimate carcinogen. Since cancer initiation is based on genotoxic event(s), we hypothesized that PCB3 and/or its metabolite(s) are mutagenic in rat liver in vivo. To investigate this, BigBlue® rats, transgenic for the lacI reporter gene, were exposed to PCB3 and 4-hydroxy-PCB3 (4-HO-PCB3). In male rats the mutant frequency (MF) of lac I in the liver was significantly elevated and the mutation spectrum differed significantly from the control. 4-HO-PCB3 caused a non-significant (p = 0.115) doubling of the MF compared to the control. These studies prove that lower halogenated biphenyls may be metabolically activated in vivo to genotoxic and initiating intermediates.

Mutagenicity of 3-methylcholanthrene, pcb3, and 4-oh-pcb3 in the lung of transgenic bigblue rats.

Recent findings of high levels of predominantly lower chlorinated biphenyls in indoor and outdoor air open the question of possible health consequences. Lower chlorinated biphenyls are more readily metabolized to reactive and potentially harmful intermediates, acting as mutagens and cancer initiators. The goal of this study was to assess the mutagenicity of PCB3 in the lungs of rats. Male BigBlue® 334 Fisher transgenic rats, which carry the bacterial lacI gene as a target of mutagenicity, were given intraperitoneal injections of corn oil, 3-methylcholanthrene (3-MC, positive control), 4-monochlorobiphenyl (PCB3) or its metabolite 4-hydroxy-PCB3 (4-OH-PCB3) weekly for 4 weeks. Lungs tissue was harvested to determine mutant frequencies, mutation spectra, and pathological changes. 3-MC caused a 15-fold increase in mutant frequency and an increase in transversion type mutations; a very early occurrence of this type of mutation in lung tissue was previously identified in Ki-ras oncogenes of lung tumors from 3-MC exposed mice. The 2-fold increase in the mutant frequency after treatment with PCB3 and 4-OH-PCB3 was not statistically significant, but a shift in the mutation spectra, especially with PCB3, and an increase in mutations outside of the hotspot region for spontaneous mutations (bp 1-400), suggest that PCB3 and possibly 4-OH-PCB3 are mutagenic in the rat lung.