Induction of chromosome aberrations in vitro by phenolphthalein: mechanistic studies.

Phenolphthalein induces tumors in rodents but because it is negative in assays for mutation in Salmonella and in mammalian cells, for DNA adducts and for DNA strand breaks, its primary mechanism does not seem to be DNA damage. Chromosome aberration (Ab) induction by phenolphthalein in vitro is associated with marked cytotoxicity. At very high doses, phenolphthalein induces weak increases in micronuclei (MN) in mouse bone marrow; a larger response is seen with chronic treatment. All this suggests genotoxicity is a secondary effect that may not occur at lower doses. In heterozygous TSG-p53((R)) mice, phenolphthalein induces lymphomas and also MN, many with kinetochores (K), implying chromosome loss. Induction of aneuploidy would be compatible with the loss of the normal p53 gene seen in the lymphomas. Here we address some of the postulated mechanisms of genotoxicity in vitro, including metabolic activation, inhibition of thymidylate synthetase, cytotoxicity, oxidative stress, DNA damage and aneuploidy. We show clearly that phenolphthalein does not require metabolic activation by S9 to induce Abs. Inhibition of thymidylate synthetase is an unlikely mechanism, since thymidine did not prevent Ab induction by phenolphthalein. Phenolphthalein dramatically inhibited DNA synthesis, in common with many non-DNA reactive chemicals that induce Abs at cytotoxic doses. Phenolphthalein strongly enhances levels of intracellular oxygen radicals (ROS). The radical scavenger DMSO suppresses phenolphthalein-induced toxicity and Abs whereas H(2)O(2) potentiates them, suggesting a role for peroxidative activation. Phenolphthalein did not produce DNA strand breaks in rat hepatocytes or DNA adducts in Chinese hamster ovary (CHO) cells. All the evidence points to an indirect mechanism for Abs that is unlikely to operate at low doses of phenolphthalein. We also found that phenolphthalein induces mitotic abnormalities and MN with kinetochores in vitro. These are also enhanced by H(2)O(2) and suppressed by DMSO. Our findings suggest that induction of Abs in vitro is a high-dose effect in oxidatively stressed cells and may thus have a threshold. There may be more than one mechanism operating in vitro and in vivo, possibly indirect genotoxicity at high doses and also chromosome loss, both of which would likely have a threshold.

TRH regulates Kv1.5 gene expression through a Galphaq-mediated PLC-independent pathway.

Thyrotropin-releasing hormone (TRH) decreases transcription of the Kv1.5 K(+) channel gene in GH(3) pituitary cells. Here, we examine whether TRH utilizes Gq activated phospholipase C, Gs or Gi to produce this response. We report that expression of constitutively active Galphaq mimicked and occluded the TRH effect. In contrast, expression of activated Galpha(S) or Galpha(i2) had no effect on Kv1. 5 mRNA expression. Furthermore, pertussis and cholera toxins failed to block the TRH-induced decrease in channel mRNA. Surprisingly, despite the role of Gq, the phospholipase C inhibitor U73122 did not alter down-regulation of channel mRNA by TRH, although it abolished the TRH-induced increase in intracellular [Ca(2+)] and up-regulation of c-fos mRNA. Furthermore, depletion of an intracellular Ca(2+) pool or inhibition of protein kinase C did not block the TRH-induced decrease in Kv1.5 mRNA. These results indicate that TRH-induced down-regulation of Kv1.5 gene expression is mediated by Galphaq proteins, but does not require PLC activation.

Comparison of mutations in the p53 and K-ras genes in lung carcinomas from smoking and nonsmoking women.

Lung cancer incidence is increasing in women with little or no tobacco exposure, and the cause of this trend is not known. One possibility is increased sensitivity to environmental tobacco smoke in women nonsmokers diagnosed with lung cancer. To determine whether mutations associated with tobacco exposure are found in the lung tumors of women who are lifetime nonsmokers or occasional smokers, we compared the p53 and K-ras mutational spectra in lung carcinomas from 23 female nonsmokers, 2 female occasional smokers (< 10 pack-years), and 30 female long-term smokers (20-100 pack-years). We also looked for p53 and K-ras mutations in three carcinoid lung tumors, two from female nonsmokers and one from a female occasional smoker. For the p53 gene, exons 4-8 were examined for mutations; for the K-ras gene, exon 1 was examined. No mutations were found in the carcinoid tumors. In lung carcinomas, p53 mutations were identified in six (26.1%) of the cases from lifetime nonsmokers and consisted of five transitions (including three C to T, one G to A, and one T to C) and one T to A transversion. In comparison, p53 mutations were identified in 10 (31.3%) of the 32 lung carcinomas from short-term and long-term smokers and consisted of six transversions (four G to T, one A to T, and one G to C), one A to G transition, one C to T transition, and two deletions of one to four bp. Mutations in the p53 gene found in nonsmokers also occurred in either different codons or different positions within a codon compared with those seen in long-term smokers. K-ras mutations in codon 12 were identified in two lung carcinomas (8.7%) from lifetime nonsmokers. The K-ras mutations found were a G to T transversion and a G to A transition. Eight (25%) of the 32 lung carcinomas from smokers contained K-ras mutations in codons 12 and 13 (four G to T transversions and four G to A transitions). In addition, six silent mutations that are most likely polymorphisms were found in both smokers and nonsmokers. These results confirm that K-ras mutations are more frequent in smokers than in nonsmokers, but that the same type of mutation in the K-ras gene is found in both groups. In contrast, although the frequency of mutation in the p53 gene was similar in lifetime nonsmokers compared with long-term smokers, the types and spectra of mutation are significantly different. Two of the C to T transitions found in nonsmokers, but none of those found in smokers, occur at the C of a CpG site. These results suggest the mutagen(s) and/or mechanisms of p53 mutations in women nonsmokers are different from those responsible for p53 mutations in women smokers, which are probably largely induced by tobacco mutagens.

Structure-activity relationships and computer-assisted analysis of respiratory sensitization potential.

The mechanism(s) underlying respiratory sensitivity to chemicals is uncertain but is assumed to involve immunologic components with pharmacologic and neurologic involvement. Predictive testing would be valuable to prevent occurrence of hypersensitivity. Several in vitro and in vivo approaches have been used for predictive purposes. In vitro methods have included assessment of the ability of the chemical to undergo reaction with proteins. Computational methods have investigated the relationship between structure and electrophilic potential of chemical allergens. We have initiated a structure-activity evaluation of chemicals associated with elicitation of respiratory sensitization and have utilized a computer-based expert system, MultiCASE. A preliminary database of 39 active chemicals has been established from a literature search of clinical case reports and animal test results. Evaluation of the model has indicated structural alerts for activity which consist of structural fragments as well as physicochemical properties. Further development of the model and evaluation of findings should enable mechanistic insight into the process of respiratory sensitization and recognition of factors which distinguish respiratory sensitizers mechanistically from other chemical allergens such as contact sensitizing chemicals.

Evaluating clinical case report data for SAR modeling of allergic contact dermatitis.

Clinical case reports can be important sources of information for alerting health professionals to the existence of possible health hazards. Isolated case reports, however, are weak evidence of causal relationships between exposure and disease because they do not provide an indication of the frequency of a particular exposure leading to a disease event. A database of chemicals causing allergic contact dermatitis (ACD) was compiled to discern structure-activity relationships. Clinical reports represented a considerable fraction of the data. Multiple Computer Automated Structure Evaluation (MultiCASE) was used to create a structure-activity model to be used in predicting the ACD activity of untested chemicals. We examined how the predictive ability of the model was influenced by including the case report data in the model. In addition, the model was used to predict the activity of chemicals identified from clinical case reports. The following results were obtained: When chemicals which were identified as dermal sensitizers by only one or two case reports were included in the model, the specificity of the model was reduced. Less than one half of these chemicals were predicted to be active by the most highly evidenced model. These chemicals possessed substructures not previously encountered by any of the models. We conclude that chemicals classified as sensitizers based on isolated clinical case reports be excluded from our model of ACD. The approach described here for evaluating activity of chemicals based on sparse evidence should be considered for use with other endpoints of toxicity when data are correspondingly limited.

Membrane depolarization inhibits Kv1.5 voltage-gated K+ channel gene transcription and protein expression in pituitary cells.

Voltage-gated K+ channels play an essential role in the production of action potential activity by excitable cells. Recent studies have suggested that expression of K+ channel genes may be regulated by stimuli that affect electrical activity. Elevating the concentration of extracellular KCl causes membrane depolarization and, thus, is widely used for studying electrical activity-dependent changes in neurons, muscle, and endocrine cells. Here we show that elevated KCl decreases Kv1.5 K+ channel mRNA expression in clonal pituitary cells without affecting Kv1.4 and Kv2.1 mRNA levels. K+ channel blockers, which cause depolarization, also produce down-regulation of Kv1.5 mRNA, while NaCl addition had no effect. Thus, the effect of KCl is mediated by K(+)-induced membrane depolarization. Unlike many known effects of K+, down-regulation of Kv1.5 mRNA does not require Ca2+ or Na+ influx, or Na(+)-H+ exchange. Furthermore, the decrease in Kv1.5 mRNA expression is due to inhibition of channel gene transcription and persists after inhibition of protein synthesis, excluding a role for induction of intermediary regulatory proteins. Finally, immunoblots with antibody specific for the Kv1.5 polypeptide show that depolarization for 8 h reduces the expression of Kv1.5 channel protein. The decrease in K+ channel protein expression caused by depolarization-induced Ca(2+)-independent inhibition of Kv1.5 gene transcription may produce a long-term enhancement of pituitary cell excitability and secretory activity.

Multiple protein kinases are required for basal Kv1.5 K+ channel gene expression in GH3 clonal pituitary cells.

The role of protein kinases in maintaining basal expression of voltage-gated K+ channel mRNA was examined in GH3 clonal pituitary cells. Nonspecific inhibition of protein kinases with H7 or staurosporine markedly decreases Kv1.5 K+ channel gene transcription and mRNA without producing a substantial change in Kv1.4 mRNA. Selective inhibitors for protein kinase C, Ca(2+)-calmodulin kinases, and tyrosine kinases do not affect Kv1.5 mRNA expression. In contrast, the Rp-diastereomer of adenosine 3',5'-cyclic monophosphorothioate, a specific inhibitor of protein kinase A, partially inhibits Kv1.5 mRNA expression (approximately 40%), and this effect was antagonized by 8-bromo-adenosine 3',5'-cyclic monophosphate. Thus, protein kinase A and at least one other kinase are required for basal Kv1.5 mRNA expression in pituitary cells.

Inhibition of voltage-gated K+ channel gene expression by the neuropeptide thyrotropin-releasing hormone.

Many neurotransmitters regulate action potential activity in neuronal, endocrine, and cardiac cells by rapidly modulating the gating of K+ channels. Neurotransmitters might also produce prolonged effects on excitability by regulating the expression of K+ channel genes. Here we show that the neuropeptide thyrotropin-releasing hormone (TRH) down-regulates Kv1.5 and Kv2.1 K+ channel mRNAs in clonal pituitary cells. The effect on Kv1.5 mRNA expression does not require protein synthesis and is due to decreased transcription. Immunoblots demonstrate that Kv1.5 and Kv2.1 immunoreactivities are significantly reduced by TRH within 12 hr. The change in channel protein expression is associated with a decrease in voltage-gated K+ currents. Thus, TRH enhances excitability by inhibiting K+ channel gene expression. Neuropeptide regulation of K+ channel gene expression may produce long-term changes in neuronal action potential activity and synaptic transmission.

Dexamethasone rapidly induces Kv1.5 K+ channel gene transcription and expression in clonal pituitary cells.

Glucocorticoids specifically increase Kv1.5 K+ channel mRNA in normal and clonal (GH3) rat pituitary cells. Here, we demonstrate that dexamethasone, a glucocorticoid agonist, rapidly induces Kv1.5 gene transcription, but does not affect Kv1.5 mRNA turnover (t1/2 approximately 0.5 hr) in GH3 cells. Immunoblots indicate that the steroid also increases the expression of the 76 kd Kv1.5 protein approximately 3-fold within 12 hr without altering its half-life (t1/2 approximately 4 hr). In contrast, Kv1.4 protein expression is unaffected. Finally, we find that the induction of Kv1.5 protein is associated with an increase in a noninactivating component of the voltage-gated K+ current. Our results indicate that hormones and neurotransmitters may act within hours to regulate excitability by controlling K+ channel gene expression.

Glucocorticoid induced up-regulation of a pituitary K+ channel mRNA in vitro and in vivo.

Hormones might produce long-term changes in cell excitability by regulating K+ channel gene expression. Recently, we found that dexamethasone increases expression of Kv1.5 K+ channel mRNA in GH3 rat pituitary tumor cells. We wished to test if this effect is specific for the Kv1.5 gene, if it is mediated by activation of glucocorticoid receptors, and whether it occurs in normal pituitary cells. Here we report that dexamethasone treatment of GH3 cells for 3 hours increases Kv1.5 mRNA without affecting Kv1.4 or Kv2.1 K+ channel mRNAs or D Ca2+ channel mRNA. Treatment with sex steroids fails to alter Kv1.5 mRNA levels, while natural glucocorticoids increase expression of the channel mRNA. RU38486, a competitive inhibitor of glucocorticoid receptors, inhibits the response to dexamethasone. We then tested whether Kv1.5 mRNA is induced by dexamethasone in normal rat pituitary cells. To study in vivo effects, channel mRNA levels in pituitaries from adrenalectomized rats were measured with RNAse protection assays. One day following dexamethasone injection Kv1.5 mRNA was increased 8-fold. Dexamethasone induction of Kv1.5 mRNA was also found in primary cultured anterior pituitary cells. We conclude that activated glucocorticoid receptors specifically induce Kv1.5 K+ channel mRNA expression in normal and clonal anterior pituitary cells.