Effects of the brominated flame retardant hexabromocyclododecane (HBCD) on dopamine-dependent behavior and brainstem auditory evoked potentials in a one-generation reproduction study in Wistar rats

Hexabromocyclododecane (HBCD) is a widely used brominated flame retardant which has been recently detected in many environmental matrices. Data from a subacute toxicity study indicated dose-related effects particularly on the pituitary thyroid-axis and retinoids in female rats. Brominated and chlorinated aromatic hydrocarbons are also reported to exert effects on the nervous system. Several investigations revealed a pronounced sensitivity of the dopaminergic system and auditory functions to polychlorinated biphenyls. Therefore, the present experiment should examine, whether or not HBCD affects these targets. Rats were exposed to 0, 0.1, 0.3, 1, 3, 10, 30 or 100 mg HBCD/kg body weight via the diet. Exposure started before mating and was continued during mating, gestation, lactation, and after weaning in offspring. Haloperidol-induced catalepsy and brainstem auditory evoked potentials (BAEPs) were used to assess dopamine-dependent behavior and hearing function in adult male and female offspring. On the catalepsy test, reduced latencies to movement onset were observed mainly in female offspring, indicating influences on dopamine-dependent behavior. The overall pattern of BAEP alterations, with increased thresholds and prolonged latencies of early waves, suggested a predominant cochlear effect. Effects were dose-dependent with lower bounds of benchmark doses (BMDL) between ≤1 and 10 mg/kg body weight for both catalepsy and BAEP thresholds. Tissue concentrations at the BMDL values obtained in this study were 3–4 orders of magnitude higher than current exposure levels in humans.     

Cytotoxicity evaluation of three pairs of hexabromocyclododecane (HBCD) enantiomers on Hep G2 cell

Hexabromocyclododecanes (HBCDs) are additive brominated flame retardants mainly used in plastics and textiles. At the present time, these compounds are found in almost all environmental and human samples. In order to evaluate the environmental safety and health risk of HBCDs, the enantiomerically pure alpha-, beta-, and gamma-HBCD were prepared using high performance liquid chromatography (HPLC) on a PM-beta-CD column and the cytotoxicities of their enantiomers were evaluated in Hep G2 cells. Results from the 3-(4,5-dimethylthioazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), resazurin reduction and lactate dehydrogenase (LDH) release assays showed a good agreement that the order of cytotoxicity was gamma-HBCDbeta-HBCD>alpha-HBCD, and that significantly lower cell viability and higher LDH release were observed in all (+)-enantiomers ((+) alpha-, (+) beta- and (+) gamma-HBCD) than the corresponding (-)-forms ((-) alpha-, (-) beta- and (-) gamma-HBCD). Additionally, the formation of reactive oxygen species (ROS) induced by these HBCD enantiomers were detected. The positive correlation between the LDH release and ROS formation demonstrated that the toxic mechanism might be mediated by oxidative damage. These results suggest that environmental and human health risks of HBCDs must be evaluated at the level of individual enantiomers.     

Impaired behaviour, learning and memory, in adult mice neonatally exposed to hexabromocyclododecane (HBCDD)

Brominated flame-retardants (BFRs) are a diverse group of global environmental pollutants. In the present study, we show that neonatal exposure to hexabromocyclododecane (HBCDD) can cause developmental behavioural defects that are similar to those recently reported for PBDEs and certain PCBs. Furthermore, HBCDD appears to be as potent as PBDEs in inducing developmental neurotoxic effects in mice.

In this study, neonatal NMRI mouse pups were given either a single oral dose of 0.9 mg HBCDD/kg body weight, 13.5 mg HBCDD/kg body weight, or a 20% fat emulsion vehicle on postnatal day 10. At the age of 3 months, the mice were observed regarding spontaneous behaviour and concerning learning and memory capability. Mice exposed to 0.9 mg HBCDD or to 13.5 mg HBCDD/kg body weight showed a significantly altered spontaneous behaviour, manifested as a hyperactive condition and reduced habituation. Learning and memory, as observed in a Morris water maze, was also significantly affected in mice given the higher dose of HBCDD.     

Neurotoxicity of the pentabrominated diphenyl ether mixture, DE-71, and hexabromocyclododecane (HBCD) in rat cerebellar granule cells in vitro

Polybrominated diphenyl ethers (PBDE) and hexabromocyclododecane (HBCD) are compounds used as additive flame retardants in plastics, electronic equipment, and textiles. The aim of the present study was to investigate the in vitro effects of the pentabrominated diphenyl ether mixture, DE-71, and HBCD on cerebellar granule cells (CGC). Both DE-71 and HBCD induced death of CGC in low micromolar concentrations. The NMDA receptor antagonist MK801 (3 μM), and the antioxidant α-tocopherol (50 μM) significantly reduced the cell death. Incubation of the compounds together with the rat liver post-mitochondrial (S9) fraction reduced cell death by 58 and 64% for DE-71 and HBCD, respectively. No ROS formation and no elevation in intracellular calcium were observed. We further demonstrated apoptotic morphology (Hoechst straining) after exposure to low levels of the two brominated flame retardants and signs of DNA laddering were found after DE-71 exposure. However, other hallmarks of apoptosis, like caspase activity, were absent indicating an atypical form of apoptosis induced by DE-71. After intraperitoneal injection of the two compounds both DE-71 and HBCD were found in significant amounts in brain (559 ± 194 and 49 ± 13 μg/kg, respectively) and liver (4,010 ± 2,437 and 1,248 ± 505 μg/kg, respectively) 72 h after injection. Our results indicate that the lower brominated PBDEs have a higher potency of bioaccumulation than HBCD, and that both compounds have a neurotoxic potential in vitro.     

Induction of hepatic enzymes and oxidative stress in Chinese rare minnow (Gobiocypris rarus) exposed to waterborne hexabromocyclododecane (HBCDD)

The objective of this study was to evaluate the sub-lethal toxicity of hexabromocyclododecane (HBCDD) in fish. Adult Chinese rare minnows as in vivo models were exposed to waterborne HBCDD from 1 to 500 microg/l for 14, 28 and 42 days. Hepatic CYP1A1 (ethoxyresorufin-O-deethylase, EROD) and CYP2B1 (pentaoxyresorufin-O-depentylase, PROD) activities were measured. At the same time, molecular biomarkers of oxidative stress were also assayed in the brain, including reactive oxygen species (ROS), lipid peroxidation products (thiobarbituric acid-reactive substances, TBARS), DNA damage and protein carbonyl, as well as superoxide dismutase (SOD) activity and glutathione (GSH) content. DNA damage was evaluated using the Comet assay on erythrocytes. Besides, the content of HBCDD in whole fish was determined after 42 days exposure. The results show that HBCDD could induce EROD and PROD at 500 microg/l after 28 days exposure, and at 100 to 500 microg/l after 42 days exposure (P<0.05), respectively. ROS formation in fish brain was observed to be increased in both time- and dose-dependent manner due to HBCDD exposure. The significant increases in TBARS and protein carbonyl contents occurred in fish brain after 28 and 42 days exposure (P<0.05). Significant DNA damage in erythrocytes by Comet assay was also found in the 100-500 microg/l exposure groups (P<0.05) after 42 days exposure. Moreover, significant depletion in brain GSH content occurred in all treated groups (P<0.05) and apparent inhibition in SOD activity in brain was observed in the groups of 10-500 microg/l concentrations during 42 days exposure. The results demonstrate that increasing duration of HBCDD exposure induced EROD and PROD activities, caused excess ROS formation, finally resulted in oxidative damage to lipids, proteins and DNA and decreased antioxidant capacities in fish. Chemical analysis of HBCDD in whole fish showed accumulation up to 654 microg/g wet weight.     

Quantification of tetrabromo benzoic acid and tetrabromo phthalic acid in rats exposed to the flame retardant Uniplex FPR-45

The first withdrawal of certain polybrominated diphenyl ethers flame retardants from the US market occurred in 2004. Since then, use of brominated non-PBDE compounds such as bis(2-ethylhexyl)-2,3,4,5-tetrabromophthalate (BEH-TEBP) and 2-ethylhexyl-2,3,4,5-tetrabromobenzoate (EH-TBB) in commercial formulations has increased. Assessing human exposure to these chemicals requires identifying metabolites that can potentially serve as their biomarkers of exposure. We administered by gavage a dose of 500 mg/Kg bw of Uniplex FRP-45 (>95 % BEH-TEBP) to nine adult female Sprague–Dawley rats. Using authentic standards and mass spectrometry, we positively identified and quantified 2,3,4,5-tetrabromo benzoic acid (TBBA) and 2,3,4,5-tetrabromo phthalic acid (TBPA) in 24-h urine samples collected 1 day after dosing the rats and in serum at necropsy, 2 days post-exposure. Interestingly, TBBA and TBPA concentrations correlated well (R² = 0.92). The levels of TBBA, a known metabolite of EH-TBB, were much higher than the levels of TBPA both in urine and serum. Because Uniplex FRP-45 was technical grade and EH-TBB was present in the formulation, TBBA likely resulted from the metabolism of EH-TBB. Taken together, our data suggest that TBBA and TBPA may serve as biomarkers of exposure to non-PBDE brominated flame retardant mixtures. Additional research can provide useful information to better understand the composition and in vivo toxicokinetics of these commercial mixtures.     

Differential gene expression profiles of human leukemia cell lines exposed to benzene and its metabolites

Benzene is a well-known environmental pollutant that can induce hematotoxicity, aplastic anemia, acute myelogenous leukemia, and lymphoma. Benzene toxicity is likely mediated through metabolites induced by means of multiple pathways. Although benzene metabolites are known to induce oxidative stress and disrupt the cell cycle, the mechanism underlying leukemogenesis is not fully understood. The aim of this study was to analyze the genome-wide expression profiles of human promyelocytic leukemia HL-60 cells that had been exposed to benzene and its metabolites. This was carried out using whole human genome oligonucleotide microarrays to ascertain potential biomarkers. Genes that were differentially expressed (>1.5-fold and p-values <0.05) after exposure to benzene (BZ), hydroquinone (HQ), and 1,4-benzoquinone (BQ) were then classified with GO, KEGG and GSEA pathway annotation. All genes that were identified were then functionally categorized as being involved in the cell cycle, the p53 signaling pathway, apoptosis, the MAPK signaling pathway, or the T cell receptor signaling pathway. Functionally important genes were further validated by means of real-time RT-PCR. The results showed that EGR1, PMAIP1, AR, CCL2, CD69, HSPA8, SLC7A11, HERPUD1, ELK1, and MKI57 genes altered their expression profiles. Similar expression profiles were also found in human erythromyeloblastoid leukemia K562 cells and in human leukemic monocyte lymphoma U937 cells. In conclusion, gene expression profiles along with GO, KEGG and GSEA pathway annotation analysis have provided an insight into the leukemogenesis as well as highlighted potential gene-based biomarkers of human leukemia cell lines when they are exposed to benzene and its metabolites.     

Cancer biology and hormesis: human tumor cell lines commonly display hormetic (biphasic) dose responses

This article assesses the nature of the dose-response relationship of human tumor cell lines with a wide range of agents including antineoplastics, toxic substances (i.e., environmental pollutants), nonneoplastic drugs, endogenous agonists, and phyto-compounds. Hormetic-like biphasic dose responses were commonly reported and demonstrated in 136 tumor cell lines from over 30 tissue types for over 120 different agents. Quantitative features of these hormetic dose responses were similar, regardless of tumor cell line or agent tested. That is, the magnitude of the responses was generally modest, with maximum stimulatory responses typically not greater than twice the control, while the width of the stimulatory concentration range was usually less than 100-fold. Particular attention was directed to possible molecular mechanisms of the biphasic nature of the dose response, as well as clinical implications in which a low concentration of chemotherapeutic agent may stimulate tumor cell proliferation. Finally, these findings further support the conclusion that hormetic dose responses are broadly generalizable, being independent of biological model, endpoint measured, and stressor agent, and represent a basic feature of biological responsiveness to chemical and physical stressors.     

Gene expression profiles of human promyelocytic leukemia cell lines exposed to volatile organic compounds

Benzene, toluene, o-xylene, ethylbenzene, trichloroethylene and dichloromethane are the most widely used volatile organic compounds (VOCs), and their toxic mechanisms are still undefined. This study analyzed the genome-wide expression profiles of human promyelocytic leukemia HL-60 cells exposed to VOCs using a 35-K whole human genome oligonucleotide microarray to ascertain potential biomarkers. Genes with a significantly increased expression levels (over 1.5-fold and p-values <0.05) with six VOCs were then classified with gene ontology and KEGG pathway annotation. At IC₂₀ doses identified genes were functionally categorized as being involved in cytokine–cytokine receptor interactions and the toll-like receptor signaling pathway, whereas exposure at IC₅₀ doses identified genes associated with the p53 signaling pathway, apoptosis, and natural killer cell-mediated cytotoxicity pathway. Functionally important immune response- and apoptosis-related genes were further validated by real-time RT-PCR. The results showed that IFIT1, IFIT2, IFIT3, USP18, INFGR2, PMAIP1, GADD45A, NFKBIA, TNFAIP3, and BIRC3 genes altered their expression profiles in a dose-dependent manner. Similar expressions profiles were also found in human erythromyeloblastoid leukemia K562 cells and in human leukemic monocyte lymphoma U937 cells. In conclusion, both gene expression profiles and gene ontology analysis have elucidated potential gene-based biomarkers and provided insights into the mechanism underlying the response of human leukemia cell lines to VOC exposure.     

Metabolism and disposition of the flame retardant tris(2,3-dibromopropyl)phosphate in the rat

The metabolism and disposition of the flame retardant, tris(2,3-dibromopropyl)phosphate (Tris-BP), were studied after po and iv administration of the 14C-labeled compound to the male rat. Tris-BP was readily absorbed from the gastrointestinal tract and rapidly distributed throughout the body. The distribution and excretion of Tris-BP derived radioactivity were similar after either po or iv administration. The only effects of route of administration on tissue distribution were slightly higher concentrations in liver after po administration and in lung after iv administration. The initial elimination of Tris-BP derived radioactivity in urine, feces, and as CO2 accounted for approximately 50% of the dose in 24 hr. An analysis of Tris-BP derived radioactivity remaining in the tissues one day after administration indicated that most of the radioactivity in all tissues was in the form of various metabolites rather than the parent compound. The terminal clearance of Tris-BP derived radioactivity from most of the tissues studied was best described by a single component exponential decay with a half-life of approximately 2.5 days. Clearance from liver and kidney was somewhat slower having a half-life of approximately 3.8 days. Approximately 33% of the radioactivity excreted in urine and approximately 50% of the radioactivity excreted in bile were identified by cochromatography with synthesized standards on high performance liquid chromatography (HPLC). Six metabolites and a trace of the parent compound were identified in urine and bile by this method. The six metabolites products of dealkylation and dehydrobromination of the parent compound. The metabolites of Tris-BP isolated from urine and bile were also formed in vitro by NADPH-dependent microsomal enzymes from rat liver. The soluble enzymes from liver metabolized Tris-BP to at least three unidentified polar metabolites.     

Nephrotoxicity of the flame retardant,tris(2,3-dibromopropyl) phosphate,and its metabolites

A single ip injection of the carcinogenic flame retardant, tris(2,3-dibromopropyl) phosphate (TRIS-BP), when administered to male Sprague-Dawley rats, caused polyuric acute renal failure with tubular necrosis involving the late proximal tubule. Glomerular filtration rate and in vitro transport of the organic acid, para-aminohippurate and the organic base, N-[¹⁴C]methylnicotinamide, were depressed. An approximately equimolar dose of the TRIS-BP metabolite, bis(2,3-dibromopropyl) phosphate (BIS-BP), caused significantly more severe renal failure. In contrast, the metabolite 2,3-dibromopropanol (BP) was nonnephrotoxic. These data suggest that TRIS-BP nephrotoxicity is mediated via its metabolite BIS-BP.     

Differential regulation of DNA repair protein Rad51 in human tumour cell lines exposed to doxorubicin

Radiotherapy and chemotherapy often induce DNA double-strand breaks in both normal and malignant cells. The proteins involved in the repair of such lesions are central to cancer prognosis and treatment, as they can be overexpressed in many cancers, accelerating malignant transformation and increasing repair capacity, potentially leading to cellular resistance. If malignant cells can be selectively targeted repair proteins could also be candidates for targeted therapy. In this study, two keyplayers in eukaryotic DNA double-strand break repair, Rad51 and DNA-dependent protein kinase catalytic subunit, were analysed in noncancerous human breast cells (MCF12A) and the breast cancer cell lines (MDA MB 231 and MCF7) in response to treatment with doxorubicin. A cell cycle-independent increase in Rad51 protein levels (a recombinase involved in homologous recombination repair) was observed 24 and 48 h after treatment in MDA MB 231 and MCF12A when exposed to low levels of doxorubicin, whereas MCF7 cells displayed a continuous decrease in Rad51 protein with increasing drug concentration. DNA-dependent protein kinase catalytic subunit, which is involved in nonhomologous end joining of DNA lesions, remained unaltered under all conditions tested. Topoisomerase II-alpha protein, the primary target of doxorubicin, was upregulated at low concentrations of doxorubicin in all cell lines tested. Here we show that Rad51 protein levels can be differentially regulated in normal and malignant breast cell lines in response to doxorubicin, independent of cell cycle state. These observations have direct relevance to chemosensitivity and add an additional prognostic factor that could be taken into account when designing targeted therapeutic regimes.     

Susceptibility to cytosine arabinoside (Ara-C)-induced cytotoxicity in human leukemia cell lines

Cytosine arabinoside (1-beta-d-arabinofuranosylcytosine; Ara-C) is the most important antimetabolite chemotherapeutic drug used for acute leukemia. We examined the difference in susceptibility to Ara-C-induced cell death among a number of typical human leukemia cell lines, NALM-6, MOLT-4, Jurkat, U937 and HL-60. NALM-6, which had a high expression level of p53, a tumor suppressor gene, was most susceptible to Ara-C. U937 and HL-60, with p53-null human leukemia cell lines were little affected by Ara-C. There was not always a correlation between susceptibility and the uptake of Ara-C. The production of reactive oxygen species (ROS) was increased in all leukemia cells. Pifithrin-alpha, a chemical inhibitor of wild-type p53, ameliorated the cytotoxicity of Ara-C in NALM-6 and MOLT-4, but not Jurkat, U937 or HL-60. Our data suggest that the mechanism of Ara-C-induced cell death is a common one, involving an increase in the production of ROS and p53-dependent cell death.     

The use of cytokine array to examine cytokine profiles of two human cell lines exposed to indoor dust

Human cytokine array was used to investigate the cytokine profile of U937 and KERTr after exposure to indoor dust or dust extracts. The release of MCP-1 was increased while release of IL-8 and IL-1β on U937 were decreased after exposure to indoor dust. The releases of RANTES, IL-8 and VEGF from KERTr after exposure to dust extract were increased. The results of IL-8 ELISA assay were consistent with the cytokine array. Real-time RT-PCR was performed to analyze relative changes in gene expression. The MCP-1 mRNA levels were increased after U937 exposure to 18 indoor dust samples, whereas, IL-8 and IL-1β mRNA level showed both up-regulation and down-regulation. The dose-related increase and decrease response was observed on MCP-1 and IL-8, respectively. Most indoor dust extracts increased RANTES, IL-8 and VEGF mRNA levels on KERTr. The dose-dependent response was observed on RANTES and IL-8. A significant correlation (r=0.48, p<0.05) was obtained between the total PAHs concentration in dust extracts and the induction of RANTES mRNA.