Health-risk assessment for workers exposed to polycyclic aromatic hydrocarbons (PAHs) in a carbon black manufacturing industry.

This study was established to assess workers' health-risks posed by PAHs exposures via both routes of inhalation and dermal contact. Personal inhalation exposure sampling was conducted on eight wet pelletizing workers and 22 packaging workers, by using a sampling train comprising an IOM personal inhalable aerosol sampler followed by an XAD-2 sorbent tube. Two workers were randomly selected from both exposure groups, and dermal exposures assessed by using soft polypropylene pads attached to the skin for nine different body surface areas for each selected worker. All personal inhalation and dermal samples were analyzed for 21 polycyclic aromatic hydrocarbon (PAH) species, and then converted to benzo[a]pyrene equivalent (BaPeq) concentrations by using the list of toxic equivalent factors (TEFs) suggested by Nisbet and LaGoy [Regul Toxicol Pharmocol 16 (1992) 290]. The resultant inhalation and dermal BaPeq exposure levels were used to estimate lifetime risks for lung cancer and skin cancer by using the BaP unit risks of 7.0 x 10(-2) (microg/m3)(-1) and 37.47(mg/kg bodyweight/day)(-1), respectively. Results show the personal inhalation BaPeq exposure levels for pelletizing and packaging workers were 622 and 774 ng/m3, respectively. The corresponding lifetime lung cancer risks estimated for both exposure groups were 4.35 x 10(-2) and 5.42 x 10(-2) respectively. For dermal exposures, results show personal dermal BaPeq exposure levels for both exposure groups were 0.664 and 0.847 microg/kg per day, respectively. The corresponding estimated lifetime skin cancer risks were 1.13 x 10(-3) and 1.56 x 10(-3), respectively. Although the estimated skin cancer risks were lower than the corresponding lung cancer risks for both exposure groups, however, both were higher than the designated significant risk level (= 10(-3)) which was defined by the US Supreme Court in 1980. Considering the bioavailability of particle-bound PAHs still remains unknown, the health risks obtained from this study could be overestimated and thus require further investigation.

Negative regulation of the antimetastatic gene Nm23-H1 by thyroid hormone receptors.

Metastasis of various malignant cells is inversely related to the abundance of the Nm23-H1 protein. The possible role of thyroid hormones in tumor metastasis has now been investigated by examining the effect of T3 on the expression of the Nm23-H1 gene. Human hepatoma HepG2 cells, in which endogenous thyroid hormone receptor subtype alpha1 (TRalpha1) is expressed at a low level, were stably transfected, either with expression plasmids encoding wild-type TRalpha1 or a dominant negative mutant of TRalpha1, or with the empty vector (yielding HepG2-Wt, HepG2-Mt, and HepG2-Neo cells, respectively). Immunoblot analysis revealed that exposure of HepG2-Wt and HepG2-Neo cells, but not HepG2-Mt cells, to T3-induced time-dependent decreases in the abundance of Nm23-H1 messenger RNA and protein, with the extent of these effects correlating with the level of expression of TRalpha1. An in vitro assay also revealed that T3 induced a marked increase in the invasive activity of HepG2-Wt cells; it induced a smaller increase in that of HepG2-Neo cells but had no effect on that of HepG2-Mt cells. Finally, the promoter region of Nm23-H1 spanning nucleotides -471 to -437 (relative to the transcriptional initiation site) inhibited the expression of a downstream reporter gene, in a T3-dependent manner, in COS-1 cells also transfected with an expression plasmid encoding TRalpha1 or TRbeta1. The DNA binding domain of TRbeta1 was required for this inhibitory effect. These results indicate that T3, acting through TRs, inhibits transcription of Nm23-H1, and that this effect is mediated by a negative regulatory element in the promoter region of the gene. Thus, it is possible that T3 promotes tumor metastasis by inducing down-regulation of Nm23-H1 expression.

Overexpression of thyroid hormone receptor beta1 is associated with thyrotropin receptor gene expression and proliferation in a human thyroid carcinoma cell line.

To correlate the differentiation phenotype of two human thyroid cancer cell lines with their expression of various molecular markers, we analyzed the mRNA levels of four thyroid-specific genes, including thyrotropin receptor (TSHR), thyroglobulin (Tg), thyroid transcription factor-1 (TTF-1), and paired-box containing transcription factor-8 (PAX-8) genes. The results showed a differentiation-status-related pattern in which a well-differentiated cell line (WRO) expressed all the four genes, in contrast to an anaplastic cell line (ARO) that expressed TTF-1 and reduced levels of TSHR, but no Tg or PAX-8 genes. Furthermore, to verify the finding of concomitant loss of beta subtype thyroid hormone receptor (TRbeta) and TSHR gene expression in neoplastic thyroid tumors (Bronnegard et al. 1994), we examined the expression levels of TRbeta1 gene in these cell lines. Whereas the WRO cells produced an abundant amount of TRbeta1 protein detectable by immunoprecipitation, the ARO cells produced none. This new observation prompted us to investigate whether overexpression of TRbeta1 protein in ARO cells might produce changes in the differentiation phenotypes. We found that the level of expression of the TSHR gene and the proliferative index of ARO cells were significantly upregulated in the cells stably transfected with wild-type TRbeta1. These findings suggest that TRbeta1 protein overexpression can affect the differentiation phenotypes and induce more efficient cell proliferation of the anaplastic ARO cells.

Expression of mutant thyroid hormone nuclear receptors in human hepatocellular carcinoma cells.

To understand the expression and role of thyroid hormone nuclear receptors (TRs) in hepatocarcinogenesis, we characterized the TRs in 16 human hepatocellular carcinoma (HCC) specimens. The full-length cDNAs for the two TR subtypes, alpha1 and beta1, were cloned from several tumors by reverse transcription-polymerase chain reaction. Southern blot analysis indicated that, in addition to the full-length cDNA, truncated TRalpha1 and TRbeta1 cDNAs were present in nine tumors (53%). In addition, point mutations detected by the mismatch RNase cleavage assay in TRalpha1 and TRbeta1 were found in 65% and 76% of the tumors, respectively. The mutations were confirmed by DNA sequencing. Interestingly, most of the TRalpha1 mutations were in amino acid codons 209-228 and 245-256, two hot-spots in HCC patients. However, no hot-spot was detected in TRbeta1. The expression of TRalpha1 and TRbeta1 proteins was determined in the tissue extracts by western blotting. TRbeta1 protein was expressed or elevated in 10 tumors but not in normal livers, whereas the expression of TRalpha1 was variable among tumors. The mutant TR proteins were translated in vitro, and their hormone- and DNA-binding activities were evaluated. Abnormal binding to the thyroid hormone response elements was observed. The proteins' DNA binding activity was either partially impaired or completely lost. The high prevalence of TR mutations found in the tumors of patients with hepatocellular carcinoma suggests that mutant TRs could play an important role in liver carcinogenesis.

Dominant negative activity of mutant thyroid hormone alpha1 receptors from patients with hepatocellular carcinoma.

Complementary DNAs for two mutant thyroid hormone alpha1 receptors (TR alpha1) were isolated from hepatocellular carcinomas of two patients. Sequence analyses of the complementary DNAs showed a single Val390Ala and double Pro398Ser/Glu350Lys mutations in mutants H and L, respectively. We characterized their hormone-binding, DNA-binding, and dominant negative activities. Mutants H and L did not bind the hormone T3. Their DNA-binding activities were analyzed using three types of thyroid hormone response elements (TREs) in which the half-site binding motifs are arranged in an everted repeat (Lys), an inverted repeat (Pal), or a direct repeat separated by four nucleotides (DR4). Compared with wild-type TR alpha1 (w-TR alpha1), which bound these TREs with different homodimer/monomer ratios, binding of mutant L to the three TREs as homodimers was reduced by approximately 90%. However, binding of mutant H to these TREs was more complex. Although it bound normally to DR4 as homodimers, its binding to Lys as homodimers was reduced by approximately 80%. Surprisingly, its binding to Pal was markedly enhanced compared with w-TR alpha1. The binding of these two mutants to the three TREs as heterodimers with retinoid X receptors (RXR alpha and -beta) was not significantly affected. Consistent with the lack of T3-binding activity, both mutants had lost their trans-activation capacity. Mutants H and L exhibited dominant negative activity, but differed in their TRE dependency. The dominant negative potency of mutant H was in the rank order of Pal > DR4 > Lys, whereas no TRE dependency was observed for mutant L. The present study indicates that mutations of the TR alpha gene do occur in patients and that these novel TR alpha1 mutants provide a valuable tool to further understand the molecular basis of the dominant negative action of mutant TRs.

Identification of naturally occurring dominant negative mutants of thyroid hormone alpha 1 and beta 1 receptors in a human hepatocellular carcinoma cell line.

To understand the function of thyroid hormone nuclear receptors (TRs) in human hepatocellular carcinoma cells (HCC), we characterized the hormone binding and transactivational activity of TRs in a HCC cell line, J7. TR alpha 1 (J7-TR alpha 1) and TR beta 1 (J7-TR beta 1) complementary DNAs were cloned from this cell line, and the binding activity to the hormone response elements (TREs) and to the thyroid hormone, 3,3',5-triiodo-L-thyronine (T3) of the expressed TR proteins were evaluated. J7-TR alpha 1 and J7-TR beta 1 bound to TREs similarly as the TRs isolated from other tissues. However, J7-TR alpha 1 did not bind to T3, and J7-TR beta 1 bound to T3 with only about 10% the affinity of the wild-type TR beta 1. Sequencing of the complementary DNAs shows a single Met259Ile mutation in J7-TR alpha 1 and Met334Val in J7-TR beta 1. Using reporters containing TREs, we found that J7-TR alpha 1 and J7-TR beta 1 had virtually lost their transactivational activity. Moreover, these two mutants inhibited the transactivational activity of the wild-type TRs by a dominant negative effect not only on the transfected TRs, but also on endogenous TRs in other two HCC cell lines, SK-Hep-1 and HepG2. The potency of the dominant negative effect of these two mutants inversely correlated with the expression level of endogenous TRs. The present studies identified two novel naturally occurring TR mutants that have potent dominant negative action. The identification of both the alpha and beta dominant negative mutants in J7 made this cell line a useful model system to further understand the molecular mechanism of the dominant negative action of TR mutants.