Evaluation of Flood Removal in Combination with Insecticide Seed Treatment for Rice Water Weevil (Coleoptera: Curculionidae) Larval Management in Rice.

An experiment was conducted at the Delta Research and Extension Center in Stoneville, MS during 2017 and 2018 to determine whether removal of the flood is an economical method of control for rice water weevil, Lissorhoptrus oryzophilus Kuschel. This experiment compared a continuous flood production system to draining a rice field completely and reestablishing a flood for the remainder of the growing season. In addition, two insecticide seed treatments, thiamethoxam and chlorantraniliprole, were compared with an untreated control within each system. Rice water weevil densities were measured prior to draining at 3 wk after flood and again after the flood was reestablished in drained plots. Rice water weevil densities were greater in 2017 than 2018. Chlorantraniliprole at the predrainage and postdrainage sample timing reduced larval numbers compared with the untreated control. The plots where water was removed until soil cracking then re-flooded had significantly lower weevil populations than plots that were continuously flooded during 2018 only. Draining of plots resulted in lower yields in 2018, but not in 2017. Additionally, both of the insecticide seed treatments resulted in greater yields and economic returns than the untreated control. Draining of flooded rice when rice water weevil larvae were present did not provide a consistent benefit, and may result in yield and economic penalties. Insecticide seed treatments consistently provided greater yield benefits in flooded rice. Based on these results, draining of flooded rice is not recommended to manage rice water weevil and insecticide seed treatments should be used to minimize economic losses.

Posttranslational modifications of p53 in replicative senescence overlapping but distinct from those induced by DNA damage.

Replicative senescence in human fibroblasts is absolutely dependent on the function of the phosphoprotein p53 and correlates with activation of p53-dependent transcription. However, no evidence for posttranslational modification of p53 in senescence has been presented, raising the possibility that changes in transcriptional activity result from upregulation of a coactivator. Using a series of antibodies with phosphorylation-sensitive epitopes, we now show that senescence is associated with major changes at putative regulatory sites in the N and C termini of p53 consistent with increased phosphorylation at serine-15, threonine-18, and serine-376 and decreased phosphorylation at serine-392. Ionizing and UV radiation generated overlapping but distinct profiles of response, with increased serine-15 phosphorylation being the only common change. These results support a direct role for p53 in signaling replicative senescence and are consistent with the generation by telomere erosion of a signal which shares some but not all of the features of DNA double-strand breaks.

Control of replicative life span in human cells: barriers to clonal expansion intermediate between M1 senescence and M2 crisis.

The accumulation of genetic abnormalities in a developing tumor is driven, at least in part, by the need to overcome inherent restraints on the replicative life span of human cells, two of which-senescence (M1) and crisis (M2)-have been well characterized. Here we describe additional barriers to clonal expansion (Mint) intermediate between M1 and M2, revealed by abrogation of tumor-suppressor gene (TSG) pathways by individual human papillomavirus type 16 (HPV16) proteins. In human fibroblasts, abrogation of p53 function by HPVE6 allowed escape from M1, followed up to 20 population doublings (PD) later by a second viable proliferation arrest state, MintE6, closely resembling M1. This occurred despite abrogation of p21(WAF1) induction but was associated with and potentially mediated by a further approximately 3-fold increase in p16(INK4a) expression compared to its level at M1. Expression of HPVE7, which targets pRb (and p21(WAF1)), also permitted clonal expansion, but this was limited predominantly by increasing cell death, resulting in a MintE7 phenotype similar to M2 but occurring after fewer PD. This was associated with, and at least partly due to, an increase in nuclear p53 content and activity, not seen in younger cells expressing E7. In a different cell type, thyroid epithelium, E7 also allowed clonal expansion terminating in a similar state to MintE7 in fibroblasts. In contrast, however, there was no evidence for a p53-regulated pathway; E6 was without effect, and the increases in p21(WAF1) expression at M1 and MintE7 were p53 independent. These data provide a model for clonal evolution by successive TSG inactivation and suggest that cell type diversity in life span regulation may determine the pattern of gene mutation in the corresponding tumors.

Conditional immortalization of human thyroid epithelial cells: a tool for analysis of oncogene action.

To overcome the difficulty of assessing oncogene action in human epithelial cell types, such as thyroid, which have limited proliferative potential in culture, we have explored the use of temperature-sensitive (ts) mutants of simian virus 40 (SV40) early region to create conditionally immortalized epithelial cell lines. Normal primary cultures of human thyroid follicular cells were transfected with a plasmid containing the SV40 early region from mutant tsA58. Expanding epithelial colonies were observed after 2 to 3 months, all of which grew to greater than 200 population doublings without crisis. All showed tight temperature dependence for growth. After switch-up to the restrictive temperature (40.5 degrees C), no further increase in cell number was seen after 1 to 2 days. However, DNA synthesis declined much more slowly; the dissociation from cell division led to marked polyploidy. Viability was maintained for up to 2 weeks. Introduction of an inducible mutant ras gene into ts thyroid cells led, as expected, to morphological transformation at the permissive temperature when ras was induced. Interestingly, this was associated with a marked reduction in net growth rate. At the restrictive temperature, induction of mutant ras caused rapid cell death. These results demonstrate the utility of a ts SV40 mutant to permit the study of oncogene action in an otherwise nonproliferative target cell and reveal important differences in the interaction between ras and SV40 T in these epithelial cells compared with previously studied cell types.

Evidence for a telomere-independent "clock" limiting RAS oncogene-driven proliferation of human thyroid epithelial cells.

An initiating role for RAS oncogene mutation in several epithelial cancers is supported by its high incidence in early-stage tumors and its ability to induce proliferation in the corresponding normal cells in vitro. Using retroviral transduction of thyroid epithelial cells as a model we ask here: (i) how mutant RAS can induce long-term proliferation in an epithelial cell in contrast to the premature senescence observed in fibroblasts; and (ii) what is the "clock" which eventually triggers spontaneous growth arrest even in epithelial clones generated by mutant RAS. The early response to RAS activation in thyroid epithelial cells showed two features not seen in fibroblasts: (i) a marked decrease in expression of the cyclin-dependent kinase inhibitor (CDKI) p27(kip1) and (ii) the absence of any induction of p21(waf1). When proliferation eventually ceased (after up to 20 population doublings) this occurred despite undiminished expression of mutant RAS and was tightly correlated with a return to the initial high level of p27(kip1) expression, together with the de novo appearance of p16(ink4a). Importantly, neither the CDKI changes nor the proliferative life span of RAS-induced epithelial clones was altered by induction of telomerase activity through forced expression of the catalytic subunit, hTERT, at levels sufficient to immortalize human fibroblasts. These data provide a basis for cell-type differences in sensitivity to RAS-induced proliferation which may explain the corresponding tumor-type specificity of RAS mutation. They also show for the first time in a primary human cell model that a telomere-independent mechanism can limit not only physiological but also oncogene-driven proliferation, pointing therefore to a tumour suppressor mechanism additional, or alternative, to the telomere clock.

Effects of Cultivars and Fungicides on Rice Sheath Blight, Yield, and Quality.

The development of sheath blight (Rhizoctonia solani)-resistant rice (Oryza sativa) cultivars will allow producers to use less fungicide and to avoid significant reductions in grain and milling yields. Among cultivars currently in cultivation in the southern United States rice-producing region, sheath blight resistance levels range from very susceptible to moderately susceptible. A study was conducted to determine the response of cultivars with different levels of susceptibility to sheath blight inoculations and fungicide applications and to determine the impact of sheath blight disease development on rice yield and quality. Sheath blight epidemics in field plots were initiated by inoculation at the panicle differentiation growth stage from 2003 through 2005. Azoxystrobin at 0.17 kg a.i. ha-1 and flutolanil at 0.56 kg a.i. ha-1 were applied in sequential applications at midboot and 50 to 70% heading. Inoculation significantly increased sheath blight severity and incidence and caused yield losses of 4% in moderately susceptible cv. Francis to 21% in very susceptible cv. Cocodrie. Milling yield was affected to a lesser extent. Fungicide treatments reduced sheath blight incidence and severity regardless of cultivar. Azoxystrobin was more effective than flutolanil in minimizing yield loss due to sheath blight in all cultivars except Francis.

Metabolism of benzo[a]pyrene on the nasal mucosa of Syrian hamsters: comparison to metabolism by other extrahepatic tissues and possible role of nasally produced metabolites in carcinogenesis.

The formation of metabolites of nasally instilled benzo[a]pyrene [(BP) CAS: 50-32-8] was determined. The study was prompted by a report that a high incidence of tumors occurred in tissues of the upper respiratory and alimentary tracts of Syrian hamsters exposed to BP aerosols. The esophagi of anesthetized hamsters were surgically catheterized so that radiolabeled material instilled as BP in the nose could be collected and analyzed for metabolites. About 50% of the instilled BP was metabolized in the nose and, potentially, would have been swallowed in an awake animal. In auxiliary experiments, homogenates of respiratory and alimentary tissues were tested for metabolic activity for BP. The nose, trachea, and lungs had about equally high activities on a per organ basis (5-7 nmol/hr), whereas all other tissues had considerably less activity. The results of the study indicate that nasal metabolism may be important in causing tumors in alimentary as well as upper respiratory tissues.

Initiation of Rice Sheath Blight Epidemics and Effect of Application Timing of Azoxystrobin on Disease Incidence, Severity, Yield, and Milling Quality.

The lack of sheath blight-resistant cultivars requires rice (Oryza sativa) farmers to use fungicides to control the disease and avoid significant reductions in grain and milling yield. Sheath blight (Rhizoctonia solani) epidemics can begin over a period of weeks during the growing season, and initiation date can have significant effects on crop damage and fungicide application timing. Studies were conducted to determine how different epidemic initiation and azoxystrobin application timings affect disease development, rice yield, and milling quality. Sheath blight epidemics in field plots were initiated by inoculation at the green ring (GR), panicle differentiation (PD), early boot (EB), and late boot (LB) growth stages in 2002 to 2004. Azoxystrobin was applied to the foliage at 0.17 kg a.i. ha-1 at 7 days after PD (PD+7), midboot (B), and 50% heading (H). Inoculation significantly increased sheath blight severity and incidence and reduced yield and milling quality. There were no significant effects of inoculation timing at the GR, PD, EB, and LB growth stages. Fungicide applications made between PD+7 and H reduced sheath blight severity and incidence, resulting in higher yield and head rice milling yield compared with inoculated but nonsprayed plots.

Some biotransformation enzymes responsible for polycyclic aromatic hydrocarbon metabolism in rat nasal turbinates: effects on enzyme activities of in vitro modifiers and intraperitoneal and inhalation exposure of rats to inducing agents.

Respiratory tract biotransformation of many xenobiotics found in inhaled environmental pollutants is generally considered essential for the mutagenic, carcinogenic, and/or toxic response of lung tissue to these xenobiotics. Typical environmental pollutants contain known carcinogens adsorbed onto particles which can deposit in the nasal pharyngeal region of the respiratory tract. The purpose of this study was to characterize the metabolic capacity of rat nasal tissue. Both oxidative and nonoxidative enzyme activities were investigated which included aryl hydrocarbon hydroxylase (AHH), epoxide hydrolase (EH), uridine 5'-diphosphate-glucuronyltransferase (UDPGT), and glutathione transferase. Specific enzyme activities of AHH, EH, UDPGT, and glutathione transferase were 0.023, 6.4, 20.4, and 24.8 nmol product per mg protein per min, respectively. Benzo(a)pyrene was metabolized by AHH to dihydrodiols, quinones, and phenols in quantities which were about 10 times greater than those reported for rat lung microsomes. Small, but detectable, quantities of benzo(a)pyrene tetrols were also measured in reaction flasks in which rat nasal tissue was incubated with benzo(a)-pyrene. Attempts to increase the microsomal enzyme activities of AHH, EH, and UDPGT by pretreating rats with various inducing agents by both i.p. injection (phenobarbital, 3-methylcholanthrene, Aroclor 1254, and 2,3,7,8-tetrachlorodibenzo-p-dioxine) and inhalation exposure (BaP) resulted in rat nasal monooxygenases only being induced (2-fold) after pretreatment with 2,3,7,8-tetrachlorodibenzo-p-dioxine. Phenobarbital increased enzyme activities of EH and UDPGT by about 50%. These data suggest that rat nasal tissue may contain multiple forms of cytochrome P-450 and of EH and UDPGT. The results from this study support the notion that nasal tissue may be important in determining the metabolic fate of inhaled xenobiotics.

Metabolism and macromolecular covalent binding of [14C]-1-nitropyrene in isolated perfused and ventilated rat lungs.

1-Nitropyrene (1-NP) originating from such sources as diesel exhaust emissions and coal combustion fly ash has been detected in the environment. 1-NP, in addition to being both a bacterial and mammalian mutagen, is carcinogenic in rats. The purpose of this study was to quantitate 1-NP metabolism and macromolecular covalent binding in the isolated perfused rat lung. Rat lungs were perfused with 2, 5, or 24 microM [14C]-1-NP for 90 min. Tidal volume and dynamic lung compliance were monitored continually throughout the perfusion to document the ventilatory pattern and the decay of tissue elasticity. Perfusate was sampled periodically throughout the duration of the experiment and analyzed for 1-NP metabolites with high-performance liquid chromatography. In all experiments, both dynamic lung compliance and tidal volume declined in a nearly linear manner and were approximately 60% of the initial value at the end of 90 min of perfusion. At all concentrations of [14C]-1-NP tested, less than 5 to 6% of the total amount of [14C]-1-NP added was metabolized in lungs from control and phenobarbital (PB)-treated rats. Lungs from control and PB- and 3-methylcholanthrene (3-MC)-treated rats metabolized [14C]-1-NP to oxidized, reduced, and conjugated metabolites. The major metabolites were 3-, 6-, and 8-hydroxynitropyrene. Small quantities of 10-hydroxynitropyrene, aminopyrene, and acetylaminopyrene were also detected in perfusates (less than 10% of the total metabolites). Treatment of rats with PB resulted in a 60% increase in the total metabolism of [14C]-1-NP, whereas treatment of rats with 3-MC resulted in a 10-fold increase in the rate of metabolism of [14C]-1-NP when compared to controls. Conjugate hydrolysis studies indicated that the water-soluble metabolites from lungs of control and PB- and 3-MC-treated rats consisted of hydroxynitropyrene glucuronides and hydroxynitropyrene sulfate conjugates. Quantities of 14C covalently bound to lung macromolecules after 90 min of perfusion from lungs of control and PB-treated rats were 0.06 to 0.21 nmol equivalents/g lung. However, in lungs from 3-MC-treated rats, there was a 20-fold increase in quantities of 14C covalently bound when compared to lungs from either control or PB-treated rats. The results from these studies point to the potential importance of lung metabolism in contributing to the metabolic fate of inhaled 1-NP.

A phenotypically and karyotypically stable human thyroid epithelial line conditionally immortalized by SV40 large T antigen.

Primary cultures of normal human neonatal thyroid follicular cells were transfected with a plasmid expressing a temperature-sensitive (tsA58) mutant of SV40 large T antigen. An epithelial cell line, designated B-thy-ts.1, was obtained which showed tight temperature-dependent growth. In sharp contrast to previous such lines, which were derived from adult thyroid, B-thy-ts.1 has retained a well-differentiated phenotype as reflected in its morphology and cytokeratin expression pattern. In addition to phenotypic stability the line also displays an unusually stable karyotype, lacking the usual clastogenic effects of SV40, which we speculate to result from a greater DNA repair capacity of its cell of origin. B-thy-ts.1 should be a particularly useful tool with which to study the effects of activated oncogenes on epithelial growth and differentiation.

Mutant p53 rescues human diploid cells from senescence without inhibiting the induction of SDI1/WAF1.

Although the cyclin-dependent kinase inhibitor p21SDI1 (WAF1/CIP1) has been proposed as the mediator of p53-induced cell cycle arrest following DNA damage, several stimuli now appear to induce SDI1 independent of p53 function. We have examined the behavior of p53 and SDI1 in an isogeneic model by manipulating p53 status in normal diploid human fibroblasts using an amphotropic retroviral vector. Following DNA strand break damage induced by bleomycin, both SDI1 induction and G1-S cell cycle arrest are p53 dependent, consistent with SDI1 being the key mediator. In contrast, in cellular senescence (and following UV irradiation), induction of SDI1 occurs independent of p53 function yet growth arrest is still p53 dependent. We conclude (a) that redundant pathways exist for induction of SDI1, but that (b) SDI1, while perhaps necessary, is not sufficient for inhibition of cell cycle progression, requiring the cooperation of an additional factor (possibly another cyclin-dependent kinase inhibitor) whose expression, at least in the case of senescence, is strictly p53 dependent.

Escape from senescence in human diploid fibroblasts induced directly by mutant p53.

Cellular senescence is thought to be a key restraint on the progression of human tumours, escape from which involves loss of function of tumour suppressor genes. The number and nature of the genes involved however is uncertain, in particular the role of p53 mutation, which is commonly correlated with tumour progression. To address this question, we used the novel approach of directly assessing the effect of mutant p53 on 'pre-aged' human diploid fibroblasts (HDF), thereby avoiding the uncertainty of additional cooperating events, inherent in transgenic models. HDF were passaged till near-senescent and then infected with an amphotropic retroviral vector encoding an ala143 human mutant p53. The results show conclusively that p53 mutation alone is sufficient to extend the proliferative lifespan of normal fibroblasts by approximately 17 population doublings, but has no phenotypic effect on 'young' fibroblasts. We conclude that a key tumour-limiting function of wild-type p53 is to mediate growth arrest after a given number of cell divisions, in agreement with data implicating a p53-regulated gene, WAF-1/sdi-1, in cellular senescence. This may be reconciled with its 'guardian of the genome' role, if telomere erosion, a key change in senescence, is perceived by the cell as a form of DNA 'damage'.

Activated ras and ret oncogenes induce over-expression of c-met (hepatocyte growth factor receptor) in human thyroid epithelial cells.

Hepatocyte Growth Factor (HGF) receptor, encoded by the protooncogene c-met, is overexpressed in many human tumours, including those of thyroid epithelium. The absence in most cases of any primary structural abnormality of the met gene suggests that overexpression is secondary to mutation of other gene(s). To test this hypothesis we investigated the effect on met expression of two activated oncogenes known to play a major role in thyroid oncogenesis, ras and ret. To minimize the possibility of unknown co-operating events, we introduced these genes directly into normal human thyrocytes in primary culture using amphotropic retroviral vectors and assessed met expression as early as possible in the resulting epithelial colonies. Double immunofluorescence revealed expression of met protein, strictly localized to cells expressing the mutant ras and ret vectors, expression in background normal cells being barely detectable. In contrast, colonies induced to proliferate at a comparable rate by a vector expressing SV40 T showed no increase in met expression. To permit quantitation by Western blotting we also extended these findings to a thyroid cell line (R18) containing a zinc-inducible mutant ras gene. Induction of the oncogene led to a fourfold increase in met protein expression. We conclude that overexpression of met is induced by activation of the ras or ret signalling pathway and not simply by deregulation of the cell cycle per se. The data suggest that the proliferative advantage conferred by these oncogenes may be in part due to the resulting sensitization of tumour epithelium to paracrine HGF secreted by stromal cells.

In vitro reconstruction of tumour initiation in a human epithelium.

Knowledge of tumour initiation in human epithelia is limited by sample availability and difficulty in experimental manipulation of human cells. The thyroid is a useful model since, in addition to multiple tumour stages, it presents two distinct 'pathways' of tumorigenesis: 'follicular' tumours, in which ras oncogene mutations occur at high frequency and 'papillary' tumours, associated with ret (or trk) activation. We have used these observations to reconstruct early thyroid tumorigenesis, using amphotropic retroviral vectors. When introduced into normal thyroid epithelial cells, mutant ras induces self-limiting growth of well-demarcated, differentiated colonies--a phenotype consistent with follicular adenoma. Activated ret on the other hand induces smaller, poorly-demarcated colonies with a morphology consistent with early papillary tumours. Mutant p53--which occurs only in the latest stages of thyroid cancer--was without effect. Our results provide the first direct experimental evidence in a human epithelium for alternative initiating oncogenes and their determination of the subsequent 'direction' of tumour development.

S phase cell-cycle arrest following DNA damage is independent of the p53/p21(WAF1) signalling pathway.

It is now likely that the cyclin-kinase inhibitor, p21(WAF1/SD11), is a key effector of p53-mediated cell-cycle arrest at the G(1)/S checkpoint following DNA damage. More recently, however, in vitro data has suggested that this pathway may also mediate the acute inhibition of DNA synthesis seen in cells already in S phase. Here we address this question in an intact cell system using normal human diploid fibroblasts in which p53 function is manipulated by expression of a dominant-negative mutant (ala(143)) introduced by a retroviral vector. Induction of DNA strand breaks in normal control fibroblasts by exposure to bleomycin led as expected to G(1)/S cell cycle arrest, induction of p2l(WAF1) and a rapid reduction in the rate of DNA synthesis in cells already in S phase. Stable expression of mutant p53 abrogated the G(1)/S (but not the G(2)/M) cell cycle checkpoint and abolished the induction of p21(WAF1), but had no significant effect on the inhibition of DNA replication in S phase nuclei. We conclude that, despite the in vitro evidence for inhibitory activity on PCNA/polymerase delta, p21(WAF1) induction does not appear to be essential for the acute inhibition of DNA synthesis in the intact cell following strand-break damage in S phase.

p53-Dependent growth arrest and altered p53-immunoreactivity following metabolic labelling with 32P ortho-phosphate in human fibroblasts.

The tumour suppressor gene p53 plays a major role in the cellular response to DNA damage, mediating growth arrest and/or apoptosis. Phosphorylation of the protein occurs at numerous sites in vivo and is likely to be a major mechanism for modulation of its activity as a transcriptional transactivator. Not surprisingly, therefore, p53 has been intensively studied by 32P metabolic labelling. Here we show however, using normal human fibroblasts, that typical labelling conditions induce (i) a p53-dependent inhibition of DNA synthesis and (ii) an increase in the cellular content of p53 protein detectable by the phosphorylation-sensitive antibody DO-1 but not by antibody DO-12. These data demonstrate for the first time that 32P labelling is sufficient to induce a biologically-significant, p53-mediated cellular response and strongly suggest that it perturbs the phosphorylation state of p53 which it is being used to measure. This highlights the need to re-evaluate earlier data by non-radioactive approaches using phospho-specific antibodies.

An amphotropic retroviral vector expressing a mutant gsp oncogene: effects on human thyroid cells in vitro.

Point mutations of the gsp protooncogene (encoding the alpha-subunit of the Gs protein) that constitutively activate the cAMP signaling pathway are a common feature of and a plausible causative mechanism for thyroid hyperfunctioning adenomas (hot nodules). To investigate the extent to which mutant gsp acting alone can induce proliferation of thyroid follicular cells, we generated an amphotropic retroviral vector (based on the pBABE-neo plasmid and psi-CRIP packaging line) to permit stable introduction of a hemagglutinin-tagged Gln227-->Leu mutant gsp gene into normal human thyrocytes in vitro. The biological activity of the vector was confirmed by detection of HA-tagged Gsp protein expression and induction of cAMP synthesis in selected target cells. Normal human thyroid follicular cells in primary monolayer culture were infected with the gsp retroviral vector or with corresponding vectors expressing mutant H-ras or neo only as positive and negative controls, respectively. Although, as before, mutant ras generated 10-20 well differentiated epithelial colonies/dish of 10(5) infected cells, with an average lifespan of 15-20 population doublings, only small groups of no more than 15-50 differentiated thyrocytes were observed with the gsp vector. In addition to standard conditions (10% FCS), infections were performed in reduced serum (1% FCS, TSH, and insulin), in the presence of isobutylylmethylxanthine, or in the presence of agents capable of closing gap junctions, with no significant difference in outcome. Although little or no proliferative response was observed regardless of the conditions, there was clear evidence of morphological response (rearrangement of the actin cytoskeleton and increased cell size). The results suggest that gsp mutation may not be a sufficient proliferogenic stimulus by itself to account for hot nodule formation.

Immortalized human pituitary cells express glycoprotein alpha-subunit and thyrotropin beta (TSH beta).

A major problem in the study of human pituitary cells is their lack of proliferative capacity in vitro. To address this issue, we have infected normal human, postmortem pituitary cells in monolayer culture with a temperature-sensitive (tsA58) mutant of SV40 large T antigen. Several epithelial-like colonies were isolated; and one, designated CHP2, has been studied in detail to identify its functional characteristics. CHP2 cells have undergone more than 150 culture passages and retain an epithelial morphology. They exhibit tight temperature-dependent growth, in the presence and absence of serum, with cell division at 33 C and growth inhibition at 39 C. CHP2 cells, at both temperatures, showed diffuse immunostaining for human alpha-subunit and focal staining for TSH beta. Gene expression was confirmed by RT-PCR and sequencing. TRH and GnRH receptors were not detectable, and their absence was confirmed by their lack of effects on intracellular calcium and inositol phospholipids. Cytogenetic analysis showed that the cells had a modal peak in the diploid range and a smaller peak in the tetraploid range. There was also a consistent loss of chromosome 22 and a normal chromosome 2 homologue, the latter being replaced by one of two chromosome 2 markers, M2A or M2B. In conclusion, we have immortalized human pituitary cells using SV40 tsT, from which we have cloned a cell line expressing alpha-subunit and TSH beta.

Metabolism and macromolecular covalent binding of benzo[a]pyrene in cultured Fischer-344 rat lung type II epithelial cells.

Pulmonary biotransformation of many xenobiotics may be important for the mutagenic, carcinogenic and/or toxic response of lung tissue to these compounds. Recently, a lung epithelial cell line (designated LEC), with morphological characteristics suggestive of type II cell origin, was developed in our laboratory. When LEC cells were co-cultivated with Chinese hamster ovary (CHO) cells in a cell-mediated mutagenesis assay, LEC metabolized promutagens to metabolites mutagenic to the CHO cells [A. P. Li, A. L. Brooks, J. M. Benson and F. F. Hahn, Environ. Mutagen. 4, 407 (1982)]. In the present investigation, rates of benzo[a]pyrene (BaP) metabolism in type II lung cells were determined, and the effects of various pollutants on rates of BaP metabolism and covalent binding of BaP to LEC macromolecules were measured. Cultures of LEC cells were incubated for 24 hr with 5 microM [14C]BaP, and the culture medium was analyzed for organic- and water-soluble metabolites. LEC cells metabolized BaP to BaP-7,8-diol and BaP-9,10-diol with total rates of formation of these metabolites measured at 500-600 pmoles per 10(6) cells per 24 hr. BaP-9,10-diol was the major metabolite accounting for about 80% of the total BaP metabolized. Enzyme hydrolysis studies revealed the presence of small quantities (less than 20% of BaP metabolized) of the glucuronide conjugates of BaP-7, 8-diol and 9-hydroxy-BaP. Pretreatment of LEC cells with benz[a]anthracene, coal gas condensate, or diesel exhaust particle extract (DEP) prior to incubation with BaP resulted in a 2- to 5-fold increase in overall rates of BaP metabolism. The largest increase in covalent binding of [14C]BaP equivalents to LEC macromolecules was seen after LEC cells were pretreated with DEP (3-fold). The data suggest that lung epithelial cells may play an important role in the biological fate of inhaled xenobiotics.