Carcinogenicity and mode of action evaluation for alpha-hexachlorocyclohexane: Implications for human health risk assessment.

Alpha-hexachlorocyclohexane (alpha-HCH) is one of eight structural isomers that have been used worldwide as insecticides. Although no longer produced or used agriculturally in the United States, exposure to HCH isomers is of continuing concern due to legacy usage and persistence in the environment. The U.S. Environmental Protection Agency (EPA) classifies alpha-HCH as a probable human carcinogen and provides a slope factor of 6.3 (mg/kg-day)(-1) for the compound, based on hepatic nodules and hepatocellular carcinomas observed in male mice and derived using a default linear approach for modeling carcinogens. EPA's evaluation, last updated in 1993, does not consider more recently available guidance that allows for the incorporation of mode of action (MOA) for determining a compound's dose-response. Contrary to the linear approach assumed by EPA, the available data indicate that alpha-HCH exhibits carcinogenicity via an MOA that yields a nonlinear, threshold dose-response. In our analysis, we conducted an MOA evaluation and dose-response analysis for alpha-HCH-induced liver carcinogenesis. We concluded that alpha-HCH causes liver tumors in rats and mice through an MOA involving increased promotion of cell growth, or mitogenesis. Based on these findings, we developed a threshold, cancer-based, reference dose (RfD) for alpha-HCH.

Involvement of TLR2 and TLR4 in inflammatory immune responses induced by fine and coarse ambient air particulate matter.

Induction of proinflammatory mediators by alveolar macrophages exposed to ambient air particulate matter has been suggested to be a key factor in the pathogenesis of inflammatory and allergic diseases in the lungs. However, receptors and mechanisms underlying these responses have not been fully elucidated. In this study, we examined whether TLR2, TLR4, and the key adaptor protein, MyD88, mediate the expression of proinflammatory cytokines and chemokines by mouse peritoneal macrophages exposed to fine and coarse PM. TLR2 deficiency blunted macrophage TNF-alpha and IL-6 expression in response to fine (PM2.5), while not affecting cytokine-inducing ability of coarse NIST Standard Reference Material (SRM 1648) particles. In contrast, TLR4(-/-) macrophages showed inhibited cytokine expression upon stimulation with NIST SRM 1648 but exhibited normal responses to PM2.5. Preincubation with polymyxin B markedly suppressed the capacity of NIST SRM 1648 to elicit TNF-alpha and IL-6, indicating endotoxin as a principal inducer of cytokine responses. Overexpression of TLR2 in TLR2/4-deficient human embryonic kidney 293 cells imparted PM2.5 sensitivity, as judged by IL-8 gene expression, whereas NIST SRM 1648, but not PM2.5 elicited IL-8 expression in 293/TLR4/MD-2 transfectants. Engagement of TLR4 by NIST SRM 1648 induced MyD88-independent expression of the chemokine RANTES, while TLR2-reactive NIST IRM PM2.5 failed to up-regulate this response. Consistent with the shared use of MyD88 by TLR2 and TLR4, cytokine responses of MyD88(-/-) macrophages to both types of air PM were significantly reduced. These data indicate differential utilization of TLR2 and TLR4 but shared use of MyD88 by fine and coarse air pollution particles.

Role of TLR4 tyrosine phosphorylation in signal transduction and endotoxin tolerance.

In this study, we examined whether tyrosine phosphorylation of the Toll-IL-1 resistance (TIR) domain of Toll-like receptor (TLR) 4 is required for signaling and blocked in endotoxin tolerance. Introduction of the P712H mutation, responsible for lipopolysaccharide (LPS) unresponsiveness of C3H/HeJ mice, into the TIR domain of constitutively active mouse DeltaTLR4 and mutation of the homologous P714 in human CD4-TLR4 rendered them signaling-incompetent and blocked TLR4 tyrosine phosphorylation. Mutations of tyrosine residues Y674A and Y680A within the TIR domains of CD4-TLR4 impaired its ability to elicit phosphorylation of p38 and JNK mitogen-activated protein kinases, IkappaB-alpha degradation, and activation of NF-kappaB and RANTES reporters. Likewise, full-length human TLR4 expressing Y674A or Y680A mutations showed suppressed capacities to mediate LPS-inducible cell activation. Signaling deficiencies of the Y674A and Y680A TLR4s correlated with altered MyD88-TLR4 interactions, increased associations with a short IRAK-1 isoform, and decreased amounts of activated IRAK-1 in complex with TLR4. Pretreatment of human embryonic kidney (HEK) 293/TLR4/MD-2 cells with protein tyrosine kinase or Src kinase inhibitors suppressed LPS-driven TLR4 tyrosine phosphorylation, p38 and NF-kappaB activation. TLR2 and TLR4 agonists induced TLR tyrosine phosphorylation in HEK293 cells overexpressing CD14, MD-2, and TLR4 or TLR2. Induction of endotoxin tolerance in HEK293/TLR4/MD-2 transfectants and in human monocytes markedly suppressed LPS-mediated TLR4 tyrosine phosphorylation and recruitment of Lyn kinase to TLR4, but did not affect TLR4-MD-2 interactions. Thus, our data demonstrate that TLR4 tyrosine phosphorylation is important for signaling and is impaired in endotoxin-tolerant cells, and suggest involvement of Lyn kinase in these processes.

TLR2- and TLR4-dependent activation of STAT1 serine phosphorylation in murine macrophages is protein kinase C-delta-independent.

Engagement of Toll-like receptor (TLR) proteins activates multiple signal transduction pathways. Previous studies demonstrated that TLR2 and TLR4 engagement leads to rapid phosphorylation of the transcription factor STAT1 at serine 727 (Ser-727 STAT1) in murine macrophages. Only TLR4 engagement induced STAT1 phosphorylation at tyrosine 701, although this response was delayed compared with Ser-727 STAT1 phosphorylation. Unlike other cell types, the p38 mitogen-activated protein kinase was necessary, but not sufficient, for TLR-induced phosphorylation of Ser-727 STAT1 in macrophages. We and others had previously shown that Ser-727 STAT1 phosphorylation could be blocked by rottlerin, an inhibitor of protein kinase C-delta (PKC-delta). Here we report that peritoneal exudate macrophages from PKC-delta-deficient mice can be activated through TLR2 and TLR4 to elicit rapid phosphorylation of Ser-727 STAT1, which was blocked by both rottlerin and the p38 inhibitor SB203580, but not by the pan-PKC inhibitor bisindoylmaleamide. Furthermore, both normal and PKC-delta-deficient macrophages secreted comparable amounts of IL-6, IP-10, and RANTES following TLR engagement. In contrast, IFN-gamma-induced STAT1 serine phosphorylation was independent of both PKC-delta and p38. Overall, these studies demonstrate that a PKC-delta-independent signaling pathway downstream of both TLR2 and TLR4 is necessary for Ser-727 STAT1 phosphorylation in primary murine macrophages.

Decreased in vitro proliferation of bladder epithelial cells from patients with interstitial cystitis.

OBJECTIVES: To determine whether explanted bladder epithelial cells from patients with interstitial cystitis (IC) display intrinsically decreased rates of proliferation in vitro, and to compare the growth rates of untreated IC and normal bladder cells with the rates of normal cells treated with a purified antiproliferative factor (APF) at levels found in urine from patients with IC. METHODS: Epithelial cell explants were prepared from the bladder biopsies of 4 patients with IC and 2 asymptomatic controls. Cell proliferation was determined by serial counting of trypan blue-negative cells. APF and mock APF were purified chromatographically, and activity was determined by (3)H-thymidine incorporation into primary normal bladder epithelial cells. Heparin-binding epidermal growth factor-like growth factor and epidermal growth factor were measured by enzyme-linked immunosorbent assay. RESULTS: Bladder epithelial cells from patients with IC proliferated significantly less than did control cells by day 2 after serum starvation (P = 0.02). Similar inhibition of the proliferation rate was seen in control cells treated with APF; APF-induced changes in heparin-binding epidermal growth factor-like growth factor, but not epidermal growth factor, production by cells were associated with changes in growth rates. CONCLUSIONS: The proliferation rate of explanted bladder epithelial cells from patients with IC in serum-free medium was significantly less than that of control cells, indicating an intrinsic abnormality in IC cell proliferation. This abnormality may be caused by APF, which induces reversible inhibition of heparin-binding epidermal growth factor-like growth factor production and normal bladder epithelial cell proliferation.

Comparison of APF activity and epithelial growth factor levels in urine from Chinese, African-American, and white American patients with interstitial cystitis.

OBJECTIVES: Interstitial cystitis (IC) is a chronic bladder disorder for which the etiology is unknown. We have previously shown that antiproliferative factor (APF), heparin-binding epidermal growth factor-like growth factor (HB-EGF), and epidermal growth factor (EGF) are sensitive and specific biomarkers for IC in white American patients. Because little is known about the epidemiology of this disorder, it is unclear whether these factors would also be useful biomarkers for IC in patients from different racial groups and/or geographic locations. METHODS: Urine specimens were collected from Chinese, white American, and African-American women with IC and age and race-matched asymptomatic normal control women. APF activity was determined by (3)H-thymidine incorporation into primary normal adult human bladder epithelial cells in vitro. The HB-EGF and EGF urine levels were determined by enzyme-linked immunosorbent assay. RESULTS: The Chinese, African-American, and white American women with IC had significantly greater urine APF activity (P <0.000001, P = 0.0008, and P <0.000001, respectively), lower urine HB-EGF levels (P <0.000001, P = 0.02, and P <0.000001, respectively), and greater urine EGF levels (P <0.000001, P = 0.002, and P <0.000001, respectively), than their matched asymptomatic controls. CONCLUSIONS: These findings confirm the utility of APF, HB-EGF, and EGF as biomarkers for IC in patients from three different racial groups and two different countries.