Single and mixture per- and polyfluoroalkyl substances accumulate in developing Northern leopard frog brains and produce complex neurotransmission alterations.

Per- and polyfluoroalkyl substances (PFAS) are present in water and >99% of human serum. They are found in brains of wildlife; however, little is known about effects on the developing brain. To determine the effects of PFAS on brain and cardiac innervation, we conducted an outdoor mesocosm experiment with Northern leopard frog larvae (Rana pipiens) exposed to control, 10 ppb perfluorooctane sulfonate (PFOS), or a PFAS mixture totaling 10 ppb that mimicked aqueous film forming foam-impacted surface water (4 ppb PFOS, 3 ppb perfluorohexane sulfonate, 1.25 ppb perfluorooctanoate, 1.25 ppb perfluorohexanoate, and 0.5 ppb perfluoro-n-pentanoate). Water was spiked with PFAS and 25 larvae (Gosner stage (GS) 25) added to each mesocosm (n = 4 mesocosms per treatment). After 30 days, we harvested eight brains per mesocosm and remaining larvae developed to GS 46 (i.e. metamorphosis) before brains and hearts were collected. Weight, length, GS, and time to metamorphosis were recorded. Brain concentrations of all five PFAS were quantified using LC/MS/MS. Dopamine and metabolites, serotonin and its metabolite, norepinephrine, γ-aminobutyric acid, and glutamate were quantified using High Performance Liquid Chromatography with electrochemical detection while acetylcholine and acetylcholinesterase activity were quantified with the Invitrogen Amplex Red Acetylcholine Assay. PFOS accumulated in the brain time- and dose-dependently. After 30 days, the mixture decreased serotonin while both PFAS treatments decreased glutamate. Interestingly, acetylcholine increased in PFAS treatments at GS 46. This research shows that developmental environmentally relevant exposure to PFAS changes neurotransmitters, especially acetylcholine.

CD200R deletion promotes a neutrophil niche for Francisella tularensis and increases infectious burden and mortality.

Pulmonary immune control is crucial for protection against pathogens. Here we identify a pathway that promotes host responses during pulmonary bacterial infection; the expression of CD200 receptor (CD200R), which is known to dampen pulmonary immune responses, promotes effective clearance of the lethal intracellular bacterium Francisella tularensis. We show that depletion of CD200R in mice increases in vitro and in vivo infectious burden. In vivo, CD200R deficiency leads to enhanced bacterial burden in neutrophils, suggesting CD200R normally limits the neutrophil niche for infection. Indeed, depletion of this neutrophil niche in CD200R-/- mice restores F. tularensis infection to levels seen in wild-type mice. Mechanistically, CD200R-deficient neutrophils display significantly reduced reactive oxygen species production (ROS), suggesting that CD200R-mediated ROS production in neutrophils is necessary for limiting F. tularensis colonisation and proliferation. Overall, our data show that CD200R promotes the antimicrobial properties of neutrophils and may represent a novel antibacterial therapeutic target.

Mitogen-activated protein kinases (MAPKs) are modulated during in vitro and in vivo infection with the intracellular bacterium Burkholderia pseudomallei.

Burkholderia pseudomallei is a Gram-negative intracellular bacterium that causes the disease melioidosis. The disease can be fatal if left untreated or when antibiotic therapy is delayed and total clearance of the pathogen from the host is often not accomplished with current therapies. Thus, new therapeutic approaches for the treatment of infections caused by B. pseudomallei are required. To better understand host responses to B. pseudomallei infection, the activation of key proteins involved in the TLR inflammatory cascade was measured by western blotting. Activation of the mitogen-activated protein kinases (MAPKs) p38 and ERK were both significantly altered during both in vitro and in vivo infection. In considering an approach for therapy of B. pseudomallei infection the inhibition of ERK was achieved in vitro using the inhibitor PD0325901, along with decreased TNF-α production. However, the reduction in phosphorylated ERK and TNF-α release did not correspond with decreased bacterial replication or enhance clearance from infected macrophages. Despite this apparent lack of effect on the intracellular growth of B. pseudomallei in vitro, it is not clear what effect inhibition of ERK activation might have on outcome of disease in vivo. It may be that decreasing the levels of TNF-α in vivo could aid in reducing the overactive immune response that is known to ensue following B. pseudomallei infection, thereby increasing host survival.

Mitogen-activated protein kinases (MAPKs) are modulated during Francisella tularensis infection, but inhibition of extracellular-signal-regulated kinases (ERKs) is of limited therapeutic benefit.

Francisella tularensis is a Gram-negative intracellular bacterium that causes the disease tularemia. The disease can be fatal if left untreated and there is currently no licenced vaccine available; the identification of new therapeutic targets is therefore required. Toll-like receptors represent an interesting target for therapeutic modulation due to their essential role in generating immune responses. In this study, we analysed the in vitro expression of the key mitogen-activated protein kinases (MAPKs) p38, JNK and ERK in murine alveolar macrophages during infection with F. tularensis. The phosphorylation profile of ERK highlighted its potential as a target for therapeutic modulation and subsequently the effect of ERK manipulation was measured in a lethal intranasal F. tularensis in vivo model of infection. The selective ERK1/2 inhibitor PD0325901 was administered orally to mice either pre- or post-challenge with F. tularensis strain LVS. Both treatment regimens selectively reduced ERK expression, but only the pre-exposure treatment produced decreased bacterial burden in the spleen and liver, which correlated with a significant reduction in the pro-inflammatory cytokines IFN-γ, MCP-1, IL-6, and TNF-α. However, no overall improvements in survival were observed for treated animals in this study. ERK may represent a useful therapeutic target where selective dampening of the immune response (to control the damaging pathology seen during infection) is combined with antibiotic treatment required to eradicate bacterial infection. This combination treatment strategy has been shown to be effective in other models of tularemia.

miR-9 Acts as an OncomiR in Prostate Cancer through Multiple Pathways That Drive Tumour Progression and Metastasis.

Identification of dysregulated microRNAs (miRNAs) in prostate cancer is critical not only for diagnosis, but also differentiation between the aggressive and indolent forms of the disease. miR-9 was identified as an oncomiR through both miRNA panel RT-qPCR as well as high-throughput sequencing analysis of the human P69 prostate cell line as compared to its highly tumorigenic and metastatic subline M12, and found to be consistently upregulated in other prostate cell lines including DU-145 and PC3. While miR-9 has been characterized as dysregulated either as an oncomiR or tumour suppressor in a variety of other cancers including breast, ovarian, and nasopharyngeal carcinomas, it has not been previously evaluated and proven as an oncomiR in prostate cancer. miR-9 was confirmed an oncomiR when found to be overexpressed in tumour tissue as compared to adjacent benign glandular epithelium through laser-capture microdissection of radical prostatectomy biopsies. Inhibition of miR-9 resulted in reduced migratory and invasive potential of the M12 cell line, and reduced tumour growth and metastases in male athymic nude mice. Analysis showed that miR-9 targets e-cadherin and suppressor of cytokine signalling 5 (SOCS5), but not NF-ĸB mRNA. Expression of these proteins was shown to be affected by modulation in expression of miR-9.

"FoxP3 Hunting" during infection with Francisella tularensis.

Francisella tularensis is a Gram-negative intracellular bacterium that can cause acute disease in mouse models of infection when administered via the inhalational route. The immune response to a pulmonary infection is typified by an initial lack of pro-inflammatory cytokines, followed by hypercytokinemia prior to host death. It remains unclear what causes this delay in the host immune response. In this study we determine the presence of FoxP3 regulatory T cells in the lung, liver and spleen following intranasal infection with F. tularensis SCHU S4. In the lung, the site of initial infection, there is an increase in FoxP3+ cells during the first few days of infection and a notable absence of these cells at the point of cytokine storm and death (day 4 post-infection). This coincides with a decrease in the anti-inflammatory cytokine TGF-ß and increases of chemokines MIP-1α, MIP-1ß and RANTES. In our model, we also observed an overall decrease in the number of regulatory T cells in the spleen, which was not as evident in the liver. Overall, this data suggests that early on in an acute F. tularensis SCHUS4 infection regulatory T cells contribute to a dampening of the pro-inflammatory response, allowing for bacterial replication and spread.

Subtype analysis of Blastocystis isolates in Swedish patients.

Blastocystis is a genetically diverse and widespread intestinal parasite of animals and humans with controversial pathogenic potential. At least nine subtypes of Blastocystis have been found in humans. The genetic diversity of Blastocystis was examined in stool samples from 68 patients from the Stockholm area, Sweden. Blastocystis was identified by light microscopy, and subtyped by sequencing the 5'-end of the small subunit ribosomal RNA gene. Five Blastocystis subtypes were identified in the 63 patients whose samples were successfully subtyped: ST1 (15.9%), ST2 (14.3%), ST3 (47.6%), ST4 (20.6%), and ST7 (1.6%). ST3 was more common in males compared to females (P=0.049). Comparative molecular analysis of Blastocystis sequences revealed intra-subtype variations within the identified subtypes with the exception of ST4. Among ST4 sequences in this study, as well as in the majority of human GenBank sequences, a limited genetic diversity was found compared to what was found among the other common subtypes (ST1, ST2 and ST3). The relative prevalence of ST4 in this study was comparable to the overall distribution of ST4 in European cohorts (16.5%). This contrasts with the sparse reports of ST4 in studies from other continents, which may indicate that the distribution of this subtype is geographically heterogeneous.

Comparison of a nontoxic variant of Clostridium perfringens α-toxin with the toxic wild-type strain.

The α-toxin produced by Clostridium perfringens is one of the best-studied examples of a toxic phospholipase C. In this study, a nontoxic mutant protein from C. perfringens strain NCTC8237 in which Thr74 is substituted by isoleucine (T74I) has been characterized and is compared with the toxic wild-type protein. Thr74 is part of an exposed loop at the proposed membrane-interfacing surface of the toxin. The mutant protein had markedly reduced cytotoxic and myotoxic activities. However, this substitution did not significantly affect the catalytic activity towards water-soluble substrate or the overall three-dimensional structure of the protein. The data support the proposed role of the 70-90 loop in the recognition of membrane phospholipids. These findings also provide key evidence in support of the hypothesis that the hydrolysis of both phosphatidylcholine and sphingomyelin are required for the cytolytic and toxic activity of phospholipases.

Novel peptide therapeutics for treatment of infections.

As antibiotic resistance increases worldwide, there is an increasing pressure to develop novel classes of antimicrobial compounds to fight infectious disease. Peptide therapeutics represent a novel class of therapeutic agents. Some, such as cationic antimicrobial peptides and peptidoglycan recognition proteins, have been identified from studies of innate immune effector mechanisms, while others are completely novel compounds generated in biological systems. Currently, only selected cationic antimicrobial peptides have been licensed, and only for topical applications. However, research using new approaches to identify novel antimicrobial peptide therapeutics, and new approaches to delivery and improving stability, will result in an increased range of peptide therapeutics available in the clinic for broader applications.

Application of high-density array-based signature-tagged mutagenesis to discover novel Yersinia virulence-associated genes.

Yersinia pestis, the causative agent of plague, and the enteropathogen Yersinia pseudotuberculosis have nearly identical nucleotide similarity yet cause markedly different diseases. To investigate this conundrum and to study Yersinia pathogenicity, we developed a high-density oligonucleotide array-based modification of signature-tagged mutagenesis (STM). Y. pseudotuberculosis YPIII mutants constructed with the tagged transposons were evaluated in the murine yersiniosis infection model. The DNA tags were amplified using biotinylated primers and hybridized to high-density oligonucleotide arrays containing DNA complementary to the tags. Comparison of the hybridization signals from input pools and output pools identified a mutant whose relative abundance was significantly reduced in the output pool. Sequence data from 31 transposon insertion regions was compared to the complete Y. pestis CO92 genome sequence. The 26 genes present in both species were found to be almost identical, but five Y. pseudotuberculosis genes identified through STM did not have counterparts in the Y. pestis genome and may contribute to the different tropisms in these closely related pathogens. Potential virulence genes identified include those involved in lipopolysaccharide biosynthesis, adhesion, phospholipase activity, iron assimilation, and gene regulation. The phospholipase A (PldA) mutant exhibited reduced phospholipase activity compared to the wild-type strain and in vivo attenuation of the mutant was confirmed. The combination of optimized double tag sequences and high-density array hybridization technology offers improved performance, efficiency, and reliability over classical STM and permits quantitative analysis of data.

Increased tumor necrosis factor-alpha production by peripheral blood leukocytes from TCDD-exposed rhesus monkeys.

Previous work has shown that exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is associated with a dose-dependent increase in the incidence and severity of endometriosis in the rhesus monkey. Studies also suggest that immune mechanisms participate in TCDD-mediated toxicity and the pathogenesis of endometriosis. Thirteen years after TCDD treatment was terminated, we characterized the phenotypic distribution of peripheral blood mononuclear cells (PBMC) from TCDD-exposed and -unexposed rhesus monkeys and determined the ability of these cells to produce cytokines and exert cytolytic activity against NK and T-cell-sensitive cell lines. We also determined whether elevated serum levels of TCDD, dioxin-like PHAH congeners, and triglycerides correlated with changes in PBMC phenotype or function. For all animals, TCDD exposure correlated with increased PBMC tumor necrosis factor-alpha (TNF-alpha) secretion in response to stimulation by T-cell mitogen and decreased cytolytic activity against NK-sensitive target cells. Furthermore, increased production of this cytokine by PHA-stimulated leukocytes was associated with elevated serum triglyceride levels. Leukocyte TNF-alpha secretion in response to viral antigen and PBMC production of interferon gamma (IFNgamma), IL-6, and IL-10 following exposure to mitogen or antigen were unaffected by previous TCDD treatment. Although TCDD exposure was not associated with changes in PBMC surface antigen expression, elevated serum concentrations of TCDD, 1,2,3,6,7,8-hexachlorodibenzofuran and 3,3',4,4',5-pentachlorobiphenyl correlated with increased numbers of CD3+/CD25- and CD3-/CD25+ leukocytes and enhanced secretion of TNF-alpha by mitogen-stimulated PBMC. These findings indicate that TCDD-exposed rhesus monkeys with endometriosis exhibit long-term alterations in systemic immunity associated with elevated serum levels of specific PHAH congeners.

Serum levels of TCDD and dioxin-like chemicals in Rhesus monkeys chronically exposed to dioxin: correlation of increased serum PCB levels with endometriosis.

Humans and animals are exposed daily to a complex mixture of polyhalogenated aromatic hydrocarbons (PHAHs). Previous work has shown that exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is associated with a dose-dependent increase in the incidence and severity of endometriosis in the rhesus monkey. Dioxin-like chemicals can also exert effects in combination with TCDD via the aryl hydrocarbon receptor. This study demonstrates that the serum levels of TCDD and specific dioxin-like PHAH congeners were increased in TCDD-treated animals with endometriosis 13 years after the TCDD exposure. Nine TCDD-exposed and 6 unexposed female rhesus monkeys were evaluated for serum content of relevant compounds and for endometriosis by surgical laparoscopy. Additional studies were done on 4 animals that died 7 to 11 years after exposure to TCDD and 4 lead-treated animals with no history of PHAH treatment. For TCDD-exposed and unexposed animals, TCDD exposure correlated with an increased serum TCDD concentration. Furthermore, TCDD exposure and an elevated serum TCDD concentration were associated with increased serum levels of triglycerides, 1,2,3,6,7,8-hexachlorodibenzofuran, 3,3',4,4'-tetrachlorobiphenyl (TCB) and 3,3'4,4',5-pentachlorobiphenyl (PnCB). Importantly, the animals with elevated serum levels of 3,3',4,4'-TCB, 3,3',4,4',5-PnCB and an increased total serum TEQ had a high prevalence of endometriosis, and the severity of disease correlated with the serum concentration of 3,3,',4,4'-TCB. Increased serum concentrations of coplanar PCBs were also present in lead-treated animals. Implications of these findings for human health and the prevalence of endometriosis in humans will be discussed.

A new cytochrome P450 (CYP30) family identified in the clam, Mercenaria mercenaria.

A full-length clone with sequence similarity to genes in the cytochrome P450 superfamily was isolated from a cDNA library prepared from female Mercenaria mercenaria gonadal tissue. This clone was isolated while screening an expression library with an antibody prepared against a peptide sequence within the ligand-binding region of the murine Ah receptor. Comparison of the predicted amino acid sequence of this clone to those of other cytochrome P450 genes indicated that the closest overall sequence similarity (38%) was to proteins predicted from genes in the CYP3 family. Northern blots indicated the presence of a transcript of the appropriate size (3.0 kb) with homology to the clam cytochrome P450. In vitro translation of the cDNA clone produced a 50.7-kDa protein as determined by SDS-polyacrylamide gel electrophoresis. The in vitro translated protein was not recognized on Western blots by two polyclonal antibodies specific for members of the CYP3 family. Since the degree of similarity to existing cytochrome P450 families was below the 40% level required for membership, and the expressed protein was not recognized by CYP3-specific antibodies, this clam cytochrome P450 cDNA has been placed in a new family, cytochrome P450 30 (CYP30).

Effects of TCDD on Ah receptor, ARNT, EGF, and TGF-alpha expression in embryonic mouse urinary tract.

Prenatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces hydronephrosis in C57BL/6N mice. The etiology of this response involves TCDD-induced hyperplasia of ureteric epithelium, which occludes the ureteric lumen, blocking the flow of urine. The present study localizes and examines the effects of TCDD on the expression of the Ah receptor (AhR), the Ah receptor nuclear translocator (ARNT), epidermal growth factor (EGF), and transforming growth factor-alpha (TGF-alpha) in the epithelial cells of the developing urinary tract, particularly the ureteric bud derivatives (ureter and tubules). Pregnant C57BL/6N mice were dosed on gestation day (GD) 10 with either corn oil or TCDD at 12 micrograms/kg; a dose of 24 micrograms/kg is expected to induce 100% hydronephrosis. The metanephric urinary tract is morphologically detectable as early as GD 12; thus, embryos were removed on GD 12, 13, and 14, and the lower dorsal torso was prepared for immunohistochemistry or in situ hybridization. Regardless of treatment, the expression of both AhR and ARNT increased in epithelial cells of the ureter and AhR increased in the metanephric tubules from GD 12-14. In situ hybridization localized the expression of AhR and ARNT mRNAs to these derivatives of the ureteric bud and levels of mRNA increased throughout the developmental period examined. There were no significant effects of TCDD treatment on expression of AhR, while TCDD significantly decreased levels of ARNT in tubules on GD 14. The epithelial cells of the ureter and tubules expressed TGF-alpha and EGF. EGF increased from GD 12 to 13 in the tubules and ureter, but there was no difference from GD 13 to 14. Treatment with TCDD reduced TGF-alpha significantly only in tubules on GD 13. TCDD exposure significantly decreased EGF in ureter and tubule cells on both GD 13 and 14. In summary, the epithelial cells of the embryonic mouse urinary tract expressed AhR, ARNT, EGF, and TGF-alpha in developmentally dependent patterns. These proteins are involved in the regulation of embryonic cell proliferation during normal urinary tract development and are probably involved in the hyperplastic response to TCDD.

Establishing guidance for the handling and containment of new chemical entities and chemical intermediates in the pharmaceutical industry.

This information is intended to guide scientists and technicians working with pharmaceutical substances early in development, before occupational exposure levels (OELs) can be set. The focus is on determining hazard categories, or occupational exposure bands, which may be applied temporarily until full health-based OEL's are in place.

Isolation and characterization of a novel gene induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in rat liver.

The differential display technique was used to identify genes whose expression was regulated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Expression of a novel sequence was up-regulated in a dose-dependent fashion in liver of Sprague-Dawley male rats exposed to both chronic and acute treatment with TCDD, as measured by densitometry of Northern blot analyses (P < 0.01). A rapid amplification of cDNA ends (RACE) procedure was used to isolate a 1.8 kb cDNA from a rat liver cDNA preparation. This cloned cDNA, called 25-Dx, was sequenced and found to encode a peptide of 223 amino acids. In control rats, the 25-Dx gene was expressed at high levels in lung and liver. A hydrophobic domain of 14 residues followed by a proline-rich domain, both located in the N-terminal region, showed 71% homology with the transmembrane domain of the precursor for the interleukin-6 receptor and a conserved consensus sequence found in the cytokine/growth factor/prolactin receptor superfamily respectively.

Estradiol stimulates tyrosine phosphorylation of the insulin-like growth factor-1 receptor and insulin receptor substrate-1 in the uterus.

The signaling pathways associated with estrogen-induced proliferation of epithelial cells in the reproductive tract have not been defined. To identify receptor tyrosine kinases that are activated in vivo by 17 beta-estradiol (E2), uteri from ovariectomized mice were examined for enhanced tyrosine phosphorylation of various receptors and a receptor substrate following treatment with this hormone. Within 4 hr after hormone exposure, extracts showed increased phosphotyrosine (P-Tyr) immunoreactivity at several bands, including 170- and 180-kDa; these bands were still apparent at 24 hr after E2. Analysis of immunoprecipitates from uterine extracts revealed that E2 enhanced tyrosine phosphorylation of the insulin-like growth factor-1 receptor (IGF-1R) and insulin receptor substrate-1 (IRS-1) by 6 hr. Comparison of supernatants from IRS-1 and control rabbit IgG immunoprecipitates indicated that the 170-kDa P-Tyr band in extracts was equivalent to IRS-1. The receptors for epidermal growth factor, platelet-derived growth factor, and basic fibroblast growth factor did not exhibit an E2-induced increase in P-Tyr content. The nonestrogenic steroid hormones examined did not stimulate the P-Tyr content of IGF-1R or IRS-1. Immunolocalization of P-Tyr and IRS-1 revealed strong reactivity in the epithelial layer of the uterus from E2-treated mice, suggesting that the majority of P-Tyr bands observed in immunoblots originate in the epithelium. Since hormonal activation of IRS-1 is epithelial, estrogen-specific, and initiated before maximal DNA synthesis occurs following treatment with hormone, this protein, as part of the IGF-1R pathway, may be important in mediating estrogen-stimulated proliferation in the uterus.

Differences in binding of epidermal growth factor to liver membranes of TCDD-resistant and TCDD-sensitive rats after a single dose of TCDD.

Epidermal growth factor (EGF) receptor has been implied as having a role in certain actions of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). After a single dose of TCDD, the receptor has been shown to be downregulated in several tissues including the liver. Two rat substrains, the Han/Wistar (Kuopio; H/W) rat and the Long-Evans (Turku AB; L-E) rat exhibit over a 1000-fold difference in their sensitivity to the lethal effect of TCDD. This large sensitivity difference was utilized in the current study to investigate whether or not a correlation exists between TCDD lethality and biochemical endpoints related to the hepatic EGF receptor. In the TCDD-sensitive L-E strain both the B(max) of the EGF receptor and the receptor protein as measured by Western blots, decreased dose and time dependently. Ten days after a lethal dose of TCDD (50 µg/kg), the downregulation was 80%. In the resistant H/W strain, two non-lethal doses were used (50 and 500 µg/kg), since the lethal dose is not known. These doses caused a downregulation already at 4 days after dosing, but no further decrease by day 10. The activity of phosphoenolpyruvate carboxykinase (PEPCK, the main gluconeogenetic enzyme in the liver and a proposed target of TCDD) decreased in H/W rats at least to the same extent as in L-E rats at both 4 and 10 days. It is concluded that EGF receptor downregulation is different in the two rat strains studied, despite the fact that a classical Ah receptor-regulated response (CYP1A1 induction) is similar. The results demonstrate that downregulation of the EGF receptor by TCDD is strain-dependent as well as dose- and time-dependent.

Induced gene transcription: implications for biomarkers.

Numerous xenobiotics regulate cellular functions by altering transcription of target genes. Use of sensitive and specific biomarkers based on gene transcript levels may help clarify the shape of the dose-response curve in the low-dose region associated with human exposures to environmental concentrations of chemicals. We have quantified gene transcription induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in both animal models and humans with the use of Northern analysis and PCR-based methods. In addition, we describe a rapid and sensitive in vitro assay that we have used to screen chemicals and biological samples for their ability to alter gene transcription. Whereas some of the endpoints in our studies such as cytochrome P-450 1A1 are predictive indicators of exposure and dose, other gene responses such as growth factors are more complex and represent a critical event, progression, or adaptation to a pathological alteration. In conclusion, measurement of toxicant-induced gene transcription will contribute to the usefulness of biomarkers in addressing issues of human health and environmentally induced disease.

Persistence of TCDD-induced hepatic cell proliferation and growth of enzyme altered foci after chronic exposure followed by cessation of treatment in DEN initiated female rats.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent tumor promoter in two-stage models of hepatocarcinogenesis. This study focuses on the persistence or reversibility of TCDD-mediated changes in livers after 30 weeks of treatment and cessation of treatment. Diethylnitrosamine (DEN) initiated animals (175 mg/kg) were promoted bi-weekly with TCDD at a dose equivalent to 125 ng/kg/day for 30 weeks without or with a following waiting period of 32 weeks before necropsy. 2,3,7,8-Tetrachlorodibenzo-p-dioxin liver concentration decreased 300-fold above background. Induction of CYP1A1 dependent enzyme activity decreased according to TCDD tissue levels. In contrast, cell proliferation, as measured by BrdU-labeling index, was still 2.8-fold increased over controls in the TCDD group with waiting period compared to a 4-fold increase over controls at the end of the 30 week dosing period. Enzyme altered hepatic foci expressing the placental form of glutathione S-transferase decreased in number but the remaining foci were significantly increased in size and the percent of liver occupied by foci was higher at the end of the waiting period as compared to livers at the end of the dosing period. Liver tumor incidence at the end of the waiting period was 71% (5 of 7 animals) and the livers showed an increase in bile duct lesions with only mild toxicity. There was pronounced bile duct proliferation in DEN/TCDD treated animals after the waiting period with intense expression of TGF alpha in bile duct epithelial cells at detected by immunohistochemical methods. In comparison, at the end of the 30 week dosing period the livers showed more severe toxicity and only mild bile duct proliferation. Also, one small hepatocellular adenoma was observed. It is concluded that as opposed to CYP1A1 induction the more complex biological responses, cell proliferation and selective growth of certain preneoplastic foci, are persistent after prolonged TCDD treatment within the experimental framework of our study.