Transcriptome correlation analysis identifies two unique craniosynostosis subtypes associated with IRS1 activation.

The discovery of causal mechanisms associated with nonsyndromic craniosynostosis has proven to be a difficult task due to the complex nature of the disease. In this study, differential transcriptome correlation analysis was used to identify two molecularly distinct subtypes of nonsyndromic craniosynostosis, termed subtype A and subtype B. In addition to unique correlation structure, subtype A was also associated with high IGF pathway expression, whereas subtype B was associated with high integrin expression. To identify a pathologic link between altered gene correlation/expression and the disease state, phosphorylation assays were performed on primary osteoblast cell lines derived from cases within subtype A or subtype B, as well as on primary osteoblast cell lines with novel IGF1R variants previously reported by our lab (Cunningham ML, Horst JA, Rieder MJ, Hing AV, Stanaway IB, Park SS, Samudrala R, Speltz ML. Am J Med Genet A 155A: 91-97, 2011). Elevated IRS1 (pan-tyr) and GSK3ß (ser-9) phosphorylation were observed in two novel IGF1R variants with receptor L domain mutations. In subtype A, a hypomineralization phenotype coupled with decreased phosphorylation of IRS1 (ser-312), p38 (thr-180/tyr-182), and p70S6K (thr-412) was observed. In subtype B, decreased phosphorylation of IRS1 (ser-312) as well as increased phosphorylation of Akt (ser-473), GSK3ß (ser-9), IGF1R (tyr-1135/tyr-1136), JNK (thr-183/tyr-187), p70S6K (thr-412), and pRPS6 (ser-235/ser-236) was observed, thus implicating the activation of IRS1-mediated Akt signaling in potentiating craniosynostosis in this subtype. Taken together, these results suggest that despite the stimulation of different pathways, activating phosphorylation patterns for IRS1 were consistent in cell lines from both subtypes and the IGF1R variants, thus implicating a key role for IRS1 in the pathogenesis of nonsyndromic craniosynostosis.

Genetic polymorphisms of microsomal and soluble epoxide hydrolase and the risk of Parkinson's disease.

Oxidative stress is hypothesized to play a major role in the destruction of dopaminergic neurons, which is associated with Parkinson's disease. Epoxides are potentially reactive intermediates formed through the oxidative metabolism of both exogenous and endogenous substances that contribute to cytotoxic damage mediated by oxidative stress. The microsomal (EPHX1) and soluble (EPHX2) epoxide hydrolases function to regulate the oxidation status of a wide range of xenobiotic- and lipid-derived substrates; therefore, interindividual variation in these pathways may mitigate epoxide-related cellular injury. In this investigation, we examined the potential association between the risk of Parkinson's disease and genetic variation within the EPHX1 and EPHX2 genes. Fluorescent 5' nuclease-based assays were developed to identify the allelic status of individuals with respect to specific single nucleotide polymorphisms in exons 3 and 4 of the EPHX1 gene and exons 8 and 13 of the EPHX2 gene. EPHX1 and EPHX2 genotype data were obtained from 133 idiopathic Parkinson's disease patients and 212 control subjects matched on age, gender and ethnicity. No statistically significant differences were found in the distribution of the reference and variant alleles between Parkinson's disease and control subjects, or when results were stratified by gender. Therefore, common polymorphisms within EPHX1 and EPHX2 do not appear to be important risk factors for Parkinson's disease.

Searching expressed sequence tag databases: discovery and confirmation of a common polymorphism in the thymidylate synthase gene.

Databases of expressed sequence tags (EST) can be used to screen rapidly for potential polymorphisms in candidate proteins. As part of this study, we screened the gene for the enzyme thymidylate synthase (TS). TS is important physiologically because it is essential for the synthesis of deoxythymidylate, a nucleotide required for DNA synthesis and repair. TS is also a major target for cancer chemotherapeutic drugs, especially the widely used 5-fluorouracil. Using sequence alignment of ESTs, we identified a candidate 6-bp variation at bp 1494 in the 3'-untranslated region of the TS mRNA. This sequence variation occurred in 21 of 34 aligned ESTs at this location, including ESTs from various tissue sources. The presence of this polymorphism was confirmed in a Caucasian population (n = 95) by polymerase chain restriction amplification/RFLP analysis. The allele frequency of the 6-bp deletion was found to be 0.29 (wildtype +6 bp/+6 bp, 48%; +6 bp/-6 bp, 44%; -6 bp/-6 bp, 7%). Although the function of this polymorphism has not yet been investigated, the 3'-untranslated region of a gene can play a role in mRNA stability and translation. This study illustrates an approach to polymorphism discovery in candidate enzymes of physiological interest by searches of publicly available sequence data, a rapid and inexpensive method. The potential functional relevance of the common 6-bp deletion in the TS gene needs to be investigated, because this enzyme is plausibly of major importance not only in cancer treatment but also in cancer prevention.

Vitamin D receptor polymorphism and the risk of colorectal adenomas: evidence of interaction with dietary vitamin D and calcium.

Laboratory studies and epidemiological investigations suggest that vitamin D plays a role in the etiology of colorectal adenomas, possibly through a mechanism mediated by the vitamin D receptor (VDR). We conducted a clinic-based case-control study to examine the association between VDR polymorphisms and colorectal adenomas. We selectively identified a random subset of 393 cases of colorectal adenomas and 406 colonoscopy-negative controls from a clinic-based case-control study conducted in the metropolitan Minneapolis/St. Paul area during 1991-1994. A self-administered questionnaire was used to collect data on dietary and supplement intake of vitamin D and calcium, as well as on demographics, physical activity, medical information, lifestyle factors, reproductive history, and anthropometry. DNA was extracted from whole blood and assayed for the BsmI VDR polymorphism using an ABI 7700 TaqMan assay. Adjusted odds ratios (OR) and 95% confidence intervals (CIs) were evaluated using logistic regression. Compared with the bb genotype (33% of controls), neither the Bb (48.8% of controls) nor the BB (18.2% of controls) genotypes was strongly associated with risk of colorectal adenomas (OR = 0.86, CI = 0.63-1.19 and OR = 0.77, CI = 0.50-1.18, respectively). However, those with the lowest tertile of vitamin D intake and the BB genotype had a lower risk of colorectal adenoma (OR = 0.24, CI = 0.08-0.76) than those with the highest tertile of intake and the bb genotype. Similarly, those with the lowest tertile of calcium intake and the BB genotype had a reduced risk of colorectal adenoma (OR = 0.34, CI = 0.11-1.06). Although it has generally been shown that higher calcium and vitamin D intake are associated with a modestly reduced risk of colorectal neoplasia, our data suggest that those with the BB BsmI VDR genotype may be at reduced risk of colorectal adenoma in the presence of lower calcium and vitamin D intake.

Assessment of regional cytochrome P450 activities in rat liver slices using resorufin substrates and fluorescence confocal laser cytometry.

Characterizing constitutive activities and inducibility of various cytochrome P450 isozymes is important for elucidating species and individual differences in susceptibility to many toxicants. Although expression of certain P450s has been studied in homogenized tissues, the ability to assess functional enzyme activity without tissue disruption would further our understanding of interactive factors that modulate P450 activities. We used precision-cut, viable rat liver slices and confocal laser cytometry to determine the regional enzyme activities of P450 isozymes in situ. Livers from control and beta-naphthoflavone (beta NF)-treated rats were sectioned with a Krumdieck tissue slicer into 250-microns thick sections. A slice perfusion chamber that mounts on the cytometer stage was developed to allow for successive measurement of region-specific P450-dependent O-dealkylation of 7-ethoxy-, 7-pentoxy-, and 7-benzyloxyresorufin (EROD, PROD, and BROD activity, respectively) in the same liver slice. Images of the accumulated fluorescent resorufin product within the tissue were acquired using a confocal laser cytometer in confocal mode. As expected, slices isolated from beta NF-treated rats showed high levels of centrilobular EROD activity compared to slices from control rats, whereas PROD and BROD activities remained at control levels. These techniques should allow for the accurate quantification of regional and cell-specific P450 enzyme activity and, with subsequent analysis of the same slice, the ability to correlate specific P450 mRNAs or other factors with enzymatic activity. Moreover, these techniques should be amenable to examination of similar phenomena in other tissues such as lung and kidney, where marked heterogeneity in cellular P450 expression patterns is also known to occur.

Quantification of multiple human cytochrome P450 mRNA molecules using competitive reverse transcriptase-PCR.

We developed a quantitative competitive reverse transcriptase-polymerase chain reaction (QC RT-PCR) assay to measure mRNA levels of seven human cytochrome P450 (P450, CYP) genes and microsomal epoxide hydrolase (EH) simultaneously. This assay employs an exogenous recombinant RNA (rcRNA) molecule as an internal standard that shares PCR primer and hybridization probe sequences with CYP1A1, CYP1A2, CYP2A6/7, CYP2D6, CYP2E1, CYP2F1, CYP3A4/5/7, and EH mRNA. Because each rcRNA molecule contains several primer sequences, an entire battery of genes that exhibit differential responsiveness to various classes of xenobiotics may be measured simultaneously from one population of cDNA molecules. In this study, we demonstrated the precision and power of the assay using small amounts of human liver total RNA. We also report for the first time quantitative profiles of P450 and EH mRNA abundance in eight human livers. Cytochrome P450 2E1 mRNA maintained the highest abundance (average 6.67 x 10(7) molecules/microg of total RNA) and least variation (13 fold) in all livers examined. Cytochrome P450 1A2, CYP2A6/7, CYP2D6, CYP3A4/5, and EH mRNAs were approximately one order of magnitude less abundant than CYP2E1 transcripts, with CYP2D6 levels exhibiting the greatest variation (220 fold) between individuals. This QC RT-PCR assay should prove valuable for measuring basal and induced mRNAs in different cell types in vitro, as well as in biomonitoring applications where individuals are exposed or hypersusceptible to certain xenobiotic-initiated toxicities.

Insulin, cortisol and catecholamines do not regulate circadian variations in fibrinolytic activity.

To evaluate possible hormonal regulators of the diurnal rhythm in fibrinolytic activity, we measured tissue plasminogen activator (t-PA) activity, plasminogen activator inhibitor activity (PAI-1), t-PA antigen, insulin, cortisol, and catecholamines in 6 healthy males (age 34 +/- 5) every 2 hours for 24 hours. Fibrinolysis was characterized by a peak in PAI-1 activity and a trough in t-PA activity at 0600 h. PAI-1 activity increased 92% and t-PA activity decreased 56% between 2400 h and 0600 h. t-PA antigen (principally a measure of t-PA/PAI-1 complex), peaked at 0800 h. In comparison, insulin levels were lowest at night when PAI-1 activity was rising. The weak inverse correlation between insulin and PAI-1 activity (r = -0.28, p less than 0.02), was not sufficient to explain the diurnal change in fibrinolysis. While cortisol demonstrated the expected circadian change, the increase in cortisol did not occur until 0400 h, 4-6 hours after the rise in PAI-1 and decrease in t-PA activity started. Supine resting plasma epinephrine and norepinephrine showed no circadian rhythm. From this data, we hypothesize that the increased level of PAI-1 in the morning consumes t-PA, leading to decreased t-PA activity and increased t-PA/PAI-1 complex. Further, we conclude that insulin, cortisol, and catecholamines are not responsible for the circadian rhythm of fibrinolysis.

Genetic polymorphisms in Parkinson's disease.

The search for genetic polymorphisms relevant to Parkinson's disease etiology and pathogenesis has been motivated by recent thinking emphasizing the potential significance of gene-environment interactions. Especially influential to this research have been the MPTP model of PD induction, hypotheses concerning oxidative stressor reactions, and epidemiological observations of an inverse relation between cigarette smoking and PD risk. This brief review summarizes trends in genetic polymorphism research, with examples provided by investigations of cytochrome P450 enzymes, monoamine oxidase, superoxide dismutase, and mitochondrial genes.

Mitochondrial ND1 sequence analysis and association of the T4216C mutation with Parkinson's disease.

Mitochondrial dysfunction originating from mutations in Complex I genes may play a role in the pathogenesis of Parkinson's disease (PD). In this study, the entire ND1 coding sequence was sequenced in 84 newly diagnosed PD cases and 127 age/gender-matched controls. Numerous missense mutations were found at low frequency (<5%), whereas a thymidine to cytosine missense mutation at position 4216 that results in the replacement of tyrosine with histidine was found in 25% of the PD case samples and in 18% of the controls. When calculated according to gender, the 4216 mutation was observed in 26% of the male cases versus 16% of male controls (Odds Ratio [OR] = 1.85; 95% CI = 0.79-4.34). In contrast, females exhibited approximately equal frequencies among cases (22.5%) and controls (21%), yielding an OR of 1.08 (95% C.I. = 0.36-3.22). The findings indicate only a weak association of this genetic variant with PD.

Tissue specific changes in the expression of glutamate-cysteine ligase mRNAs in mice exposed to methylmercury.

Glutamate-cysteine ligase (GLCL), the rate-limiting enzyme in glutathione (GSH) synthesis is composed of two subunits, a catalytic (GLCLc) and a regulatory subunit (GLCLr). These two subunits are known to be differentially regulated in vitro, in different cell types and in response to various xenobiotic exposures. In this study, we examined whether these two subunits can also be differentially regulated in vivo. We found that GLCLc and GLCLr are differentially regulated at the transcriptional level in a tissue-dependent manner in female mice treated with methylmercury (MeHg). MeHg caused a downregulation of both subunit mRNAs in the liver, upregulation of both subunit mRNAs in the kidney and upregulation of only the catalytic subunit mRNA in the small intestine of female mice treated with a single dose of MeHg (6 mg/kg) by intraperitoneal injection. These results suggest that GLCLc and GLCLr can be differentially regulated in vivo, and that this regulation is tissue dependent in the mouse.

Human glomerular cells in vitro: isolation and characterization.

Human glomerular cells were isolated, identified, and propagated in vitro, and a number of features were found to be unique to glomerular visceral epithelial and mesangial cells. Epithelial cells are identified by their "epithelial" morphology in vitro, growth pattern, presence of C3b surface receptors, response to mitogens, synthesis of a single collagen type (IV), and nearly exclusive synthesis of one type of sulfated glycosaminoglycan, heparan sulfate. Mesangial cells differ from epithelial cells and other potential contaminating cells, such as fibroblasts or endothelial cells, by their characteristic light and electron microscopic morphology and their synthesis of specific collagen and glycosaminoglycan types. In addition, while they closely resemble vascular smooth muscle cells in many ways, they differ with respect to their response to mitogens. It should now be possible to study these cells for other features, including the presence of alloantigens. This property and their response to inflammatory mediators or cells are prerequisites to the determination of their role in the pathogenesis of renal allograft rejection.

Genetic risk factors for major bleeding in patients treated with warfarin in a community setting.

The influence of warfarin pharmacogenomics on major bleeding risk has been little studied in long-term users and non-specialist care settings. We conducted a case-control study to evaluate associations between CYP2C9*2/*3, VKORC1(1173), and CYP4F2*3 variants and major bleeding among patients treated with warfarin in a community setting. We calculated major bleeding odds ratios, adjusting for race, duration of warfarin use, age, gender, and body mass index. In 265 cases and 305 controls with 3.4 and 3.7 mean years of warfarin use, respectively, CYP4F2*3 was associated with decreased major bleeding risk (odds ratio: 0.62; 95% confidence interval: 0.43-0.91). CYP2C9*2/*3 and VKORC1(1173) had null associations overall, but there was a nonsignificant increase in major bleeding risk in patients with duration <6 months (odds ratio: 1.30; 95% confidence interval: 0.60-2.83; odds ratio: 1.23; 95% confidence interval: 0.57-2.64, respectively). In summary, in the largest study of warfarin pharmacogenomics and major bleeding to date, we found a 38% lower risk in patients with CYP4F2*3, potentially reflecting interaction with warfarin and dietary vitamin K intake and warranting additional evaluation.

The effect of nicotinamide on gene expression in a traumatic brain injury model.

Microarray-based transcriptional profiling was used to determine the effect of nicotinamide on gene expression in an experimental traumatic brain injury (TBI) model. Ingenuity Pathway Analysis (IPA) was used to evaluate the effect on relevant functional categories and canonical pathways. At 24 h, 72 h, and 7 days, respectively, 70, 58, and 76%, of the differentially expressed genes were up-regulated in the vehicle treated compared to the sham animals. At 24 h post-TBI, there were 150 differentially expressed genes in the nicotinamide treated animals compared to vehicle; the majority (82%) down-regulated. IPA analysis identified a significant effect of nicotinamide on the functional categories of cellular movement, cell-to-cell-signaling, antigen presentation and cellular compromise, function, and maintenance and cell death. The canonical pathways identified were signaling pathways primarily involved with the inflammatory process. At 72 h post-cortical contusion injury, there were 119 differentially expressed genes in the nicotinamide treated animals compared to vehicle; the majority (90%) was up-regulated. IPA analysis identified a significant effect of nicotinamide on cell signaling pathways involving neurotransmitters, neuropeptides, growth factors, and ion channels with little to no effect on inflammatory pathways. At 7 days post-TBI, there were only five differentially expressed genes with nicotinamide treatment compared to vehicle. Overall, the effect of nicotinamide on counteracting the effect of TBI resulted in significantly decreased number of genes differentially expressed by TBI. In conclusion, the mechanism of the effect of nicotinamide on secondary injury pathways involves effects on inflammatory response, signaling pathways, and cell death.

Functional variants of the lipoprotein lipase gene and the risk of preeclampsia among non-Hispanic Caucasian women.

Hypertriglyceridemia is an important pathophysiologic feature of preeclampsia, a common vascular disorder of pregnancy. Three well-documented functional variants (N291S, S447X, and D9N) of the lipoprotein lipase gene were related to hypertriglyceridemia. Results from the only two studies concerning the relationship between these variants and preeclampsia risk have been inconsistent. We investigated this relationship in a case-control study including 144 preeclamptic and 290 normotensive pregnant women (all non-Hispanic Caucasians). We estimated odds ratios (OR) and 95% confidence intervals (CI) adjusted for maternal age, pre-pregnancy body mass index, and parity. After adjusting for covariates, women with the 291 N/S or S/S genotype had significantly increased risk of preeclampsia (OR 6.9, 95% CI 1.9-25.4) compared with women with the common 291N/N genotype. The 447 S/X or X/X genotype was not significantly associated with preeclampsia risk. The frequency of the 9N variant allele was 1.8% in controls, while this allele was not observed among cases. Haplotype 9D/291S/447S was strongly associated with higher risk of preeclampsia as compared with the most common haplotype 9D/291N/447S (adjusted OR 6.6, 95% CI 1.7-25.0). Results from our study support the thesis that abnormal lipid metabolism is important in the pathogenesis of preeclampsia.

Regiospecific expression of cytochrome P-450s and microsomal epoxide hydrolase in human brain tissue.

The central nervous system is an important potential target for certain environmental protoxins, but relatively little is known regarding brain-specific expression of biotransformation enzyme systems. We undertook the present study to identify regional and cellular expression patterns of individual cytochrome P-450 genes (CYP) and microsomal epoxide hydrolase (mEH) in human brain. Various regions of normal human brain were isolated and examined with respect to mRNA levels of CYP1A1, CYP1A2, CYP2E1, CPY3A, and mEH, using specific oligomer probes and reverse transcriptase-coupled polymerase chain reaction analysis. We also used immunohistochemical techniques, with antipeptide-derived antibodies, to identify specific cells from various regions of the human brain producing CYP1A1 and mEH protein. Relatively equivalent mRNA expression levels of mEH were detected in the cerebellum (C), frontal (F), occipital (O), pons (P), red nucleus (RN), and substantia nigra (SN) regions of brain. The mRNA expression patterns of CYP2E1 and CYP1A2 were similar; although detected in all brain regions examined, the RN and SN exhibited lower levels of CYP2E1 and CYP1A2 mRNA expression compared to other regions. In addition, regional differences in CYP3A and CYP1A1 mRNA expression also were observed, with the highest level of CYP3A mRNA present in the P region compared to the C, F, O, and RN, while no CYP3A mRNA was detected in the SN. CYP1A1 mRNA expression was evident in all brain regions, but the levels of CYP1A1 mRNA in the P and RN were lower than in the C, F, O, and SN. In all cases, the regional mRNA expression levels of these CYP and mEH mRNAs were less than the corresponding levels detected from the same individual's liver. CYP1A1 and mEH immunoreactivity was present in most neurons of the SN, RN, P, median raphae, locus ceruleus, inferior vestibular nucleus, dorsal motor nucleus of the vagus, and thalamus. Some but not all astrocytes within these regions also demonstrated 1A1 and mEH immunoreactivity. These results indicate that many neurons and astrocytes express mEH and CYP1A1 as well as other CYP genes, and suggest that localized biotransformation events within the certain central nervous system may account for toxicities initiated by exposure to certain environmental chemicals.

Influence of extracellular matrix overlay on phenobarbital-mediated induction of CYP2B1, 2B2, and 3A1 genes in primary adult rat hepatocyte culture.

To obtain efficient induction by phenobarbital (PB) of cytochrome P450 (CYP) genes in primary hepatocyte culture, previous studies have demonstrated the importance of culturing hepatocytes on a substratum derived from extracellular matrix (ECM or Matrigel), or in highly enriched medium formulations such as Chee's. In the present study, we have reexamined a variety of hepatocyte culture conditions and their relative abilities in preserving the PB-induction response within the CYP2B and 3A gene subfamilies. A modified culture system was developed that combines a highly effective medium formulation in conjunction with dilute concentrations of a Matrigel overlay. Specifically, hepatocytes were attached to a Collagen I substratum and overlaid with either daily (50 micrograms/ml medium) or single (233 micrograms/ml) additions of Matrigel. The PB-induction response obtained in vitro closely resembled that occurring in vivo. Induction in culture by 1 mM PB of CYP2B1, 2B2, and 3A1 mRNA levels was highly dependent on a variety of factors, including medium formulation and 0.1 microM dexamethasone addition. The response to dexamethasone addition on this gene battery varied in a medium-specific manner. Cells maintained a higher level of PB-induction response when maintained in Williams' E medium than with Chee's, Waymouth's, or Ultraculture media. The kinetics of PB induction also were more rapid in cells cultured in Williams' E medium. PB exposures in Chee's medium resulted in elevation of two CYP2B-immunoreactive proteins as detected by Western blot analysis, together with increased rates of O-dealkylation of benzyloxy- and pentoxyresorufin. However, Chee's formulation produced an abnormal PB-induced expression of CYP1A1, as determined by mRNA analysis, high rates of O-dealkylation of 7-ethoxyresorufin, and inhibition of enzymatic activity by 1 microM alpha-naphthoflavone. This paradoxical expression of CYP1A1 was not observed in PB-treated cultures grown in Williams' E medium. Thus, these studies demonstrated that the use of a physiologically balanced medium, i.e., Williams' E formulation, together with an overlay of ECM, preserves PB-induction responsiveness closely resembling that observed in vivo, and should better facilitate mechanistic investigations into the molecular nature of PB induction.

Expression of cytochrome P450s and microsomal epoxide hydrolase in primary cultures of human umbilical vein endothelial cells.

In view of the potential role of the cytochrome P450 (CYP) and microsomal epoxide hydrolase (mEH) biotransformation enzymes in the metabolism of protoxicants in the circulatory system, we examined CYP and mEH expression in several primary cultures of human umbilical vein endothelial cells (HUVEC), each established from a different individual. Total RNA was isolated from untreated cells and cells 72 hr after exposure to dimethyl sulfoxide (DMSO), Arochlor 1254 (PCB), and beta-naphthoflavone (beta NF). Specific mRNA transcripts were examined by Northern blotting and reverse transcriptase-coupled polymerase chain reaction (RT/PCR) analyses. CYP2E1, CYP3A, and CYP1A2 mRNAs were not detectable in any of the cultures by Northern blot analysis with radiolabeled oligomer probes; however, CYP1A1 mRNA was detected using this procedure in HUVEC cultures exposed to beta NF for 72 hr. Using RT/PCR, constitutive levels of CYP1A1, CYP1A2, CYP2E1, and CYP3A gene expression in HUVEC cultures were evident; however, constitutive CYP2B6 mRNA was not detected. Constitutive CYP1A2 transcript levels were detected in four of six HUVEC cultures, but levels varied between individual cultures. CYP1A2 mRNA levels were also increased in HUVEC cultures exposed to PCB and beta NF. No increases in the levels of CYP2E1 and CYP3A mRNAs were observed in HUVEC cells subsequent to PCB or beta NF exposures. Constitutive CYP2E1 transcript levels were present in all HUVEC cultures examined and varied among individuals. All HUVEC cultures examined for mEH activity exhibited constitutive levels of mEH which varied 40% between individual cultures and produced on average, 1.51 pmol benzo[a]pyrene 4,5-dihydrodiol per milligram protein per minute of reaction. Thus, these results demonstrate that human endothelial cells express CYP and mEH gene products and suggest that these enzymes may play important roles in determining metabolic fates for circulating protoxicants.

Granular pneumocytes in primary culture secrete several major components of the extracellular matrix.

Primary cultures of rat alveolar type II epithelial cells (granular pneumocytes) produced several components of the pulmonary extracellular matrix. Fractionation by ion-exchange chromatography of radiolabeled protein secreted into the culture medium resulted in the partial purification of two of these components: fibronectin and type IV procollagen. Identification of these proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was confirmed by radioimmune-precipitation studies with affinity-purified antibodies. Thrombospondin, a platelet alpha-granule protein that was recently shown to be secreted by endothelial and other mesenchymally derived cells and may be involved in platelet aggregation, was, in addition, purified by elution from diethylaminoethylcellulose with 0.5 M NaCl. The levels of these secreted proteins were measured by radioimmune precipitation. Of the total radiolabeled culture medium protein secreted during a 24-h period by the granular pneumocytes, fibronectin, type IV procollagen, and thrombospondin represented 3-15%, 2%, and 3%, respectively. The biosynthesis, by alveolar epithelial cells, of proteins that constitute or are closely associated with the alveolar basement membrane implies that this structure is at least partially derived from the cells themselves. Furthermore, it suggests that the type II epithelial cell is involved in pulmonary cytodifferentiation, in lung morphogenesis and repair, and in certain interstitial lung disorders in which derangement of the extracellular matrix occurs.

Effect of Tissue-Culture Substratum and Extracellular Matrix Overlay on Liver-Selective and Xenobiotic Inducible Gene Expression in Primary Rat Hepatocytes.

In a previous study (Sidhu et al., 1993), we demonstrated that a combination of certain cell culture media, hormone addition, and extracellular matrix (ECM) overlay coordinately modulated the expression of certain liver-selective genes in primary rat hepatocyte cultures, including the responsiveness of genes to phenobarbital. However, little is known about the interactions between the type of substratum upon which hepatocytes are adhered and the ECM overlay, as codeterminants of liver-selective gene expression. The present study was undertaken to compare specific substrata, including tissue culture-grade plastic, Primaria, and type 1 collagen-coated plastic, in combination with the presence or absence of standard ECM or a growth-factor-reduced ECM overlay. Hepatocyte cultures were assessed either as control cultures or subsequent to treatment for 24 h with phenobarbital (0.1 or 1 mM), or beta-naphthoflavone (22 µM), to monitor responses of hepatocytes to two prototypic gene-inducing agents. Analyses of maintenance and induction of cytochrome P450 and liver-selective gene expression included measures of mRNA levels using Northern blot and slot-blot hybridization and single cell immunofluorescence assays to measure levels of specific cytochrome P450 proteins. The results of these experiments demonstrated that hepatocyte-selective expression, including the absolute level of induction response (relative to those observed in the rat liver in vivo) was highly dependent on the presence of ECM overlay but independent of the substratum employed. As studied herein, the establishment of optimal conditions for primary hepatocyte culture, enabling reproduction of responses observed in vivo, is important to further prospects for in vitro toxicity testing and for investigating molecular mechanisms of phenobarbital-mediated gene regulation.

Expression of cytochrome P450 and microsomal epoxide hydrolase in cervical and oral epithelial cells immortalized by human papillomavirus type 16 E6/E7 genes.

Epidemiological evidence suggests that the presence of human papillomaviruses (HPV), when combined with smoking behaviors, considerably enhances the risk of developing oral, cervical, vulvar, and/or anal carcinomas. It is well established that the cytochrome P450 (CYP), microsomal epoxide hydrolase (mEH), and other biotransformation enzymes are important modulators of the bioactivation and detoxification of many environmental chemicals, including constituents of tobacco smoke such as certain nitrosamines and polycyclic aromatic hydrocarbons (PAH). Since there is little information regarding oral and cervical epithelial-specific expression of these genes, established primary and HPV-immortalized oral and cervical epithelial cell lines were analyzed for morphology, mRNA and protein expression patterns of specific CYPs and mEH. Primary human oral and cervical epithelial cells were immortalized using retroviral infection with HPV-16 E6/E7 genes. Primary human keratinocyte cells were immortalized by transfection of HPV-18 and made tumorigenic with nitrosomethylurea treatment. Expression profiles for mEH, CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2D6, CYP3A, and CYP2E1 were evaluated in these cultures in the presence or absence of a PAH inducer, using reverse transcriptase-coupled polymerase chain reaction analysis. mEH gene expression was evident in all cultures, while CYP2A6 mRNA was not detected in any of the cell lines, regardless of culture conditions. CYP2E1 mRNA expression was greatest in the oral epithelial cultures and detectable in all other epithelial cultures except for the HPV-18 immortalized keratinocyte cell line. Elevated levels of CYP2D6 mRNA existed in both oral epithelial cell lines and the HPV-16 immortalized cervical epithelial cells when compared to the other cell lines examined. CYP1A1 and CYP1A2 mRNAs were detected in all the cells and several cultures were inducible by PAH exposure. To corroborate the RT/PCR data, Western immunoblotting experiments were conducted on selected samples. Using these methods, CYP1A1 and CYP2E1 proteins were detected in primary and HPV-immortalized oral and cervical epithelial cultures. These data indicate that both primary and HPV immortalized cells appear to express certain biotransformation enzymes necessary for the activation of tobacco-specific nitrosamines and PAHs. Although the overall impact of HPV gene infection on expression of these systems remains to be fully elucidated, as in vitro system is characterized which should prove useful in examining interactive mechanisms of HPV with xenobiotic activation in the etiology of squamous cell carcinomas.