Subcortical serotonin 5HT2c receptor-containing neurons sex-specifically regulate binge-like alcohol consumption, social, and arousal behaviors in mice.

Binge alcohol consumption induces discrete social and arousal disturbances in human populations that promote increased drinking and accelerate the progression of Alcohol Use Disorder. Here, we show in a mouse model that binge alcohol consumption disrupts social recognition in females and potentiates sensorimotor arousal in males. These negative behavioral outcomes were associated with sex-specific adaptations in serotonergic signaling systems within the lateral habenula (LHb) and the bed nucleus of the stria terminalis (BNST), particularly those related to the receptor 5HT2c. While both BNST and LHb neurons expressing this receptor display potentiated activation following binge alcohol consumption, the primary causal mechanism underlying the effects of alcohol on social and arousal behaviors appears to be excessive activation of LHb5HT2c neurons. These findings may have valuable implications for the development of sex-specific treatments for mood and alcohol use disorders targeting the brain's serotonin system.

TAAC - TMS Adaptable Auditory Control: A universal tool to mask TMS clicks.

BACKGROUND: Coupling transcranial magnetic stimulation with electroencephalography (TMS-EEG) allows recording the EEG response to a direct, non-invasive cortical perturbation. However, obtaining a genuine TMS-evoked EEG potential requires controlling for several confounds, among which a main source is represented by the auditory evoked potentials (AEPs) associated to the TMS discharge noise (TMS click). This contaminating factor can be in principle prevented by playing a masking noise through earphones. NEW METHOD: Here we release TMS Adaptable Auditory Control (TAAC), a highly flexible, open-source, Matlab®-based interface that generates in real-time customized masking noises. TAAC creates noises starting from the stimulator-specific TMS click and tailors them to fit the individual, subject-specific click perception by mixing and manipulating the standard noises in both time and frequency domains. RESULTS: We showed that TAAC allows us to provide standard as well as customized noises able to effectively and safely mask the TMS click. COMPARISON WITH EXISTING METHODS: Here, we showcased two customized noises by comparing them to two standard noises previously used in the TMS literature (i.e., a white noise and a noise generated from the stimulator-specific TMS click only). For each, we quantified the Sound Pressure Level (SPL; measured by a Head and Torso Simulator - HATS) required to mask the TMS click in a population of 20 healthy subjects. Both customized noises were effective at safe (according to OSHA and NIOSH safety guidelines) and lower SPLs with respect to standard noises. CONCLUSIONS: At odds with previous methods, TAAC allows creating effective and safe masking noises specifically tailored on each TMS device and subject. The combination of TAAC with tools for the real-time visualization of TEPs can help control the influence of auditory confounds also in non-compliant patients. Finally, TAAC is a highly flexible and open-source tool, so it can be further extended to meet different experimental requirements.

Production and purification of higher molecular weight chondroitin by metabolically engineered Escherichia coli K4 strains.

The capsular polysaccharide obtained from Escherichia coli K4 is a glycosaminoglycan-like molecule, similar to chondroitin sulphate, that has established applications in the biomedical field. Recent efforts focused on the development of strategies to increase K4 polysaccharide fermentation titers up to technologically attractive levels, but an aspect that has not been investigated so far, is how changes in the molecular machinery that produces this biopolymer affect its molecular weight. In this work, we took advantage of recombinant E. coli K4 strains that overproduce capsular polysaccharide, to study whether the inferred pathway modifications also influenced the size of the produced polymer. Fed-batch fermentations were performed up to the 22 L scale, in potentially industrially applicable conditions, and a purification protocol that allows in particular the recovery of high molecular weight unsulphated chondroitin, was developed next. This approach allowed to determine the molecular weight of the purified polysaccharide, demonstrating that kfoF overexpression increased polymer size up to 133 kDa. Higher polysaccharide titers and size were also correlated to increased concentrations of UDP-GlcA and decreased concentrations of UDP-GalNAc during growth. These results are interesting also in view of novel potential applications of higher molecular weight chondroitin and chondroitin sulphate in the biomedical field.

Molecular, Morphological, and Functional Characterization of Corticotropin-Releasing Factor Receptor 1-Expressing Neurons in the Central Nucleus of the Amygdala.

The central nucleus of the amygdala (CeA) is a brain region implicated in anxiety, stress-related disorders and the reinforcing effects of drugs of abuse. Corticotropin-releasing factor (CRF, Crh) acting at cognate type 1 receptors (CRF1, Crhr1) modulates inhibitory and excitatory synaptic transmission in the CeA. Here, we used CRF1:GFP reporter mice to characterize the morphological, neurochemical and electrophysiological properties of CRF1-expressing (CRF1+) and CRF1-non-expressing (CRF1-) neurons in the CeA. We assessed these two neuronal populations for distinctions in the expression of GABAergic subpopulation markers and neuropeptides, dendritic spine density and morphology, and excitatory transmission. We observed that CeA CRF1+ neurons are GABAergic but do not segregate with calbindin (CB), calretinin (CR), parvalbumin (PV), or protein kinase C-δ (PKCδ). Among the neuropeptides analyzed, Penk and Sst had the highest percentage of co-expression with Crhr1 in both the medial and lateral CeA subdivisions. Additionally, CeA CRF1+ neurons had a lower density of dendritic spines, which was offset by a higher proportion of mature spines compared to neighboring CRF1- neurons. Accordingly, there was no difference in basal spontaneous glutamatergic transmission between the two populations. Application of CRF increased overall vesicular glutamate release onto both CRF1+ and CRF1- neurons and does not affect amplitude or kinetics of EPSCs in either population. These novel data highlight important differences in the neurochemical make-up and morphology of CRF1+ compared to CRF1- neurons, which may have important implications for the transduction of CRF signaling in the CeA.

The ω-SQUIPT as a tool to phase-engineer Josephson topological materials.

Multi-terminal superconducting Josephson junctions based on the proximity effect offer the opportunity to tailor non-trivial quantum states in nanoscale weak links. These structures can realize exotic topologies in several dimensions, for example, artificial topological superconductors that are able to support Majorana bound states, and pave the way to emerging quantum technologies and future quantum information schemes. Here we report the realization of a three-terminal Josephson interferometer based on a proximized nanosized weak link. Our tunnelling spectroscopy measurements reveal transitions between gapped (that is, insulating) and gapless (conducting) states that are controlled by the phase configuration of the three superconducting leads connected to the junction. We demonstrate the topological nature of these transitions: a gapless state necessarily occurs between two gapped states of different topological indices, in much the same way that the interface between two insulators of different topologies is necessarily conducting. The topological numbers that characterize such gapped states are given by superconducting phase windings over the two loops that form the Josephson interferometer. As these gapped states cannot be transformed to one another continuously without passing through a gapless condition, they are topologically protected. The same behaviour is found for all of the points of the weak link, confirming that this topology is a non-local property. Our observation of the gapless state is pivotal for enabling phase engineering of different and more sophisticated artificial topological materials.

Protection of human keratinocyte mtDNA by low-level nitric oxide.

This study was designed to evaluate the DNA damaging effects of nitric oxide and to determine whether the endogenous generation of nitric oxide at low levels in the cell exerts a protective effect against this damage. Damage to mitochondrial and nuclear DNA in normal human epidermal keratinocytes (NHEK) was assessed after treatment of these cells with varying concentrations of S-nitroso-N-acetylpenicillamine, which decomposes to release nitric oxide. The results showed that mitochondrial DNA was more vulnerable to nitric oxide-induced damage than was a similarly sized fragment of the beta-globin gene. To evaluate the effects on DNA damage by pretreatment of cells with low-levels of nitric oxide, NHEK cells were treated with the prodrug V-PYRRO/NO. This agent is metabolized inside these cells and releases small quantities of nitric oxide. The cells then were exposed to damaging amounts of nitric oxide produced by S-nitroso-N-acetylpenicillamine. The results of these studies showed that pretreatment of NHEK cells with V-PYRRO/NO attenuated the mtDNA damage and loss of cell viability produced by exposure to S-nitroso-N-acetylpenicillamine.

Mechanisms of nitric oxide-induced cytotoxicity in normal human hepatocytes.

Chronic exposure of hepatocytes to reactive nitrogen species (RNS) following liver injury and inflammation leads not only to functional and morphological alterations in the liver but also to degenerative liver diseases and hepatocellular carcinoma. Previously, we showed that S-nitroso-N-acetylpenicillamine-amine (SNAP), which generates nitric oxide, and 3-morpholinosydnonimine (Sin-1), which generates equal molar concentrations of superoxide and nitric oxide resulting in peroxynitrite production, exhibited different levels of cytotoxicity to normal human hepatocytes in culture. The aim of the present study was to elucidate some of the molecular and cellular pathways leading to hepatocyte cell death induced by RNS. Following treatment of the hepatocytes with SNAP or Sin-1, gene-specific DNA damage was measured in mtDNA and a hprt gene fragment using a quantitative Southern blot analysis. Both agents induced dose-dependent increases in DNA damage that was alkaline labile, but not sensitive to both formamidopyrimidine-DNA glycosylase (fpg) and endonuclease III, which recognize 8-oxoguanine, thymine glycol, and other oxidized pyrimidines. DNA damage was two- to fivefold greater in mtDNA than in the hprt gene fragment. There was a persistent and marked increase in DNA damage posttreatment that appeared to arise from the disruption of electron transport in the mitochondria, generating reactive species that saturated the repair system. DNA damage induced by Sin-1 and SNAP led to cell-cycle arrest in the S-phase, growth inhibition, and apoptosis. The data support the hypothesis that the functional and morphological changes observed in liver following chronic exposure to RNS are, in part, the result of persistent mitochondrial and nuclear DNA damage.

Modulation of Ki67, p53 and RARbeta expression in normal, premalignant and malignant human oral epithelial cells by chemopreventive agents.

BACKGROUND: Aberrant expression of Ki67, p53 and RARbeta are characteristic of many tumor types including those of the oral cavity. Chemopreventive agents may act by modulating their expression to more normal levels. MATERIALS AND METHODS: The effects of 21 chemopreventive agents on the expression of Ki67, p53 and RARbeta were determined using a human in vitro model of normal, premalignant and malignant oral epithelial cell lines. RESULTS: Ki67 and mutant p53 (mtp53) were overexpressed in both the premalignant and malignant cell lines, whereas expression of RARbeta was high in the normal, low in the premalignant and not detectable in the malignant cell lines. Most of the agents selectively inhibited the expression of Ki67 in the premalignant and malignant cell lines. Eight of the 21 agents increased, while four agents decreased, the levels of mtp53 protein in the premalignant cell line. In the malignant cell line, five of the agents increased, while ten agents decreased mtp53 protein levels. The agents increased RARbeta expression to near normal levels in the premalignant cell line. CONCLUSION: The data suggest that the suppression of Ki67 and mtp53 are good indicators of the effectiveness of agents in premalignant and malignant oral cells, whereas the enhancement of RARbeta is a measure of effectiveness in premalignant oral cells.

Differential response of normal, premalignant and malignant human oral epithelial cells to growth inhibition by chemopreventive agents.

Squamous cell carcinoma (SCC) of the oral cavity is a multistep process, progressing through a series of discrete, irreversible and complementary alterations in genes that control cell growth, death, and differentiation. In the premalignant state, the oral mucosa progresses through various grades of epithelial dysplasia, with the potential to convert to SCC. Chemopreventive strategies are designed to suppress, reverse, or prevent the formation of premalignant lesions and their subsequent progression to SCC. In the present study, we determined the growth inhibitory effect of 21 chemopreventive agents in a cell culture model using normal, premalignant, and malignant human oral mucosal cell lines. There were significant differences in the growth inhibitory responses of these cell lines to selected retinoids and non-retinoid analogs. Among the retinoids tested, the synthetic retinamides, as a class, showed selective growth inhibition of both premalignant and malignant cells compared to normal human oral epithelial cells in culture. Within the retinamide class, 2CPR exhibited the greatest selectivity in the growth inhibition of premalignant and malignant cells. Among the non-retinoids analyzed, DFMO was a moderate to potent inhibitor of malignant and premalignant oral cell growth, respectively, and stimulated normal oral cell growth at low concentrations. Using this in vitro approach, we have identified several potential chemopreventive agents for oral cancer as selective growth inhibitors of premalignant ahd malignant human oral mucosa cells.

Enhanced expression of the 8-oxo-7,8-dihydrodeoxyguanosine triphosphatase gene in human breast tumor cells.

The expression of the 8-oxo-7,8-dihydrodeoxyguanosine triphosphatase (8-oxo-dGTPase) gene in human breast tumors was evaluated at the level of the single cell to better understand how breast tumor cells regulate expression in response to oxidative stress. Compared to normal breast ductal cells, the level of 8-oxo-dGTPase expression in the breast tumor cells increased from non-detectable levels in normal breast to expression in 30-85% of the tumor cells in individual tumors. There was no significant association between 8-oxo-dGTPase expression and tumor grade and metastatic malignancy. The upregulation of 8-oxo-dGTPase was not directly linked to the expression of cyclins D1 and D3, estrogen receptor, p53, Ki-67 and c-erbB-2, which are genes involved in cell cycle regulation and tumor growth. The elevated expression of 8-oxo-dGTPase in human breast ductal carcinoma cells appears to be a general characteristic of breast tumors and may provide the tumor cell with a cellular defense mechanism to prevent the incorporation of 8-hydroxy-deoxyguanosine during DNA replication.

Expression of molecular biomarkers in primary breast tumors implanted into a surrogate host: increased levels of cyclins correlate with tumor progression.

BACKGROUND: The overexpression or amplification of tumor suppressor and proto-oncogenes are important factors in the progression of breast cancer. Recent attention has focused on the cyclin genes, whose involvement in signal transduction pathways regulate cell cycle progression. The amplification of the cyclins D1 and D3 genes usually leads to loss of normal growth control and is thought to play an important growth regulatory role in tumor development and progression. In this report, we investigate the association of altered cyclin expression with other prognostic indicators (histological grade, lymph node status, estrogen receptor, p53, and c-erbB-2) in the progression of human breast cancer. MATERIALS AND METHODS: Surgical tumor specimens were obtained from 16 breast tubular ductal, and invasive ductal carcinomas and grafted onto gnotobiotic nude (nu/nu) mice. The expression diversity and distribution of the localization of the protein products of the c-erbB-2, cyclins D1 and D3, p53, and estrogen receptor were characterized immunohistochemically and the results in the original tumor (T0) were compared with those in the tumors that developed in nude mice (T1) xenografts. RESULTS: The T0 tumors exhibited a diversity of cellular morphology in the tumor matrix and diversity in expression of these proteins. These specific changes were also preserved in the T1 tumors. Whereas 67% of the T1 tumors exhibited high numbers of estrogen receptorpositive nuclei, only 50% of these tumors grew when grafted onto nude mice. The histological grade (14/15 were G2 to G3) and metastatic malignancy in the lymph nodes (10/15) did not appear to be related to tumor growth in the nude mouse. There was no relationship between those tumors which exhibited high percentages of c-erbB-2- and p53-positive cells and growth in nude mice. A strong association (p < 0.001) was observed between the overexpression of cyclin D1 transcripts in the T0 tumors and the continued growth of the T1 tumors in nude mice. In the T1 tumors, both cyclins D1 and D3, estrogen receptor, and p53 were observed in 49% to 86% of the cells of the T1 tumors examined; the number of cells expressing c-erbB-2 protein varied widely in these tumors. CONCLUSIONS: The results indicate that the tumor matrix exhibits a diversity in the level of phenotypic expression of genes involved in cellular growth of breast tumors in both the T0 or T1 host environment. Changes in cyclin activity appear to correlate with the vigorous level of breast tumor growth and progression.

Sensitivity of human hepatocytes in culture to reactive nitrogen intermediates.

The cytotoxic effects of 3-morpholinosydnonimine (Sin-1) and S-nitroso-N-acetylpenicillamine-amine (SNAP) on replicatively active human hepatocyte cells in culture was determined as a function of oxidant type. Both Sin-1 which yields nitric oxide and peroxynitrite following the generation of superoxide anion plus nitric oxide, and SNAP which generates nitric oxide, induced dose dependent decreases in the colony forming capabilities of the human hepatocytes. Sin-1 was much more cytotoxic (LD50 = 400 microM) than SNAP (LD50 = 1250 microM). Comparatively, both compounds were much less cytotoxic than H2O2 (LD50 = 96 microM). Sin-1 induced 4-fold higher levels of cellular nitrite than that generated by the chemical in cell free medium. Nitrotyrosine, a marker of peroxynitrite formation in cells, was immunohistochemically detected in hepatocytes treated with both Sin-1 and SNAP. The formation of 3-nitrotyrosine by hepatocytes incubated with SNAP, suggests that hepatocytes generate intracellular superoxide which reacts with the exogenous nitric oxide derived from SNAP to produce intracellular peroxynitrite, resulting in the SNAP cytotoxicity. The enhanced levels of Sin-1 cytotoxicity on the hepatocytes is suggested to be due both to the chemical generation of peroxynitrite and superoxide anion by Sin-1. These data indicate that peroxynitrite is formed in cultured human hepatocytes inhibiting their replication, and that peroxynitirite may play a significant role in the pathogenesis of liver disease.

Mechanism of enhancement of esophageal tumorigenesis by 6-phenylhexyl isothiocyanate.

6-Phenylhexyl isothiocyanate (PHITC) enhances esophageal tumorigenesis induced by the carcinogen N-nitrosomethylbenzylamine (NMBA) in rats while its shorter chain analog, phenethyl isothiocyanate (PEITC), inhibits NMBA-induced esophageal tumorigenesis. A significant increase in O6-methylguanine levels in esophageal DNA at 72 h after NMBA administration to rats pretreated with PHITC suggested that PHITC might enhance NMBA metabolic activation or inhibit DNA repair. To test this hypothesis, groups of 20 rats were administered PEITC or PHITC at concentrations of 0, 1.0, or 2.5 mmol/kg in modified AIN-76A diet for 2 weeks. The esophagi were removed from rats, stripped, split, and maintained in HEPES buffered saline (HBS) for assays of NMBA metabolism (n = 5 per group) or were snap frozen for DNA repair assays (n = 15 per group). The principal metabolites of NMBA produced by esophageal explants were: two unidentified peaks, benzyl alcohol (at 4 h only), and benzoic acid. Esophageal explants from PEITC-treated animals showed a significantly decreased ability to metabolize NMBA as expected. PHITC-treated animals showed a slight inhibition in the formation of most NMBA-related metabolites, rather than an overall increase in NMBA activation. This inhibition was less than that observed with PEITC. No inhibitory effects were observed on O6-alkylguanine transferase (AGT) activity in the esophagi of rats treated with 1.0 micromol/g or 2.5 micromol/g PHITC. Thus, effects of PHITC on esophageal metabolism and DNA repair do not account for the enhancement of NMBA tumorigenicity by PHITC.

Molecular characterization of mutations in the hprt gene of normal human skin keratinocytes treated with N-ethyl-N-nitrosourea: influence of O6-alkylguanine alkyltransferase.

O6-Alkylguanine-DNA alkyltransferase (AGT) is responsible for repairing the O6-alkylguanine lesion in DNA. There is wide variation in the levels of AGT between organ and cell types, which appears to correlate with cell and tissue type sensitivity to the mutagenic and carcinogenic effects of alkylating agents. In order to investigate the role of AGT in modulating the frequency and types of mutations induced in one type of normal human parenchymal cells, we examined the types and frequency of mutations in the hypoxanthine (guanine) phosphoribosyltransferase (hprt gene in 116 mutants derived from two N-ethyl-N-nitrosourea (ENU)-treated normal human skin keratinocyte cell lines. O6-Benzylguanine (O6-BZ; 5 microM x 2 hours) was used to specifically inhibit AGT activity before ENU treatment (0 to 5 mM x 1 hour). O6-BZ increased both the cytotoxic and mutagenic effects of ENU by 1.8- and 3- to 5-fold, respectively. In both treatment groups, most of the mutations were base substitutions (72%). The proportion of GC to AT transitions in the O6-BZ group (14/31) was twice that in the group treated with ENU alone, consistent with the loss of AGT activity in these cells. There was no strand specificity of GC to AT and AT to GC transitions in both groups. Base transversions accounted for 28% of total base substitutions. A lower than expected proportion of AT to TA transversions were observed in both cell lines, which decreased in the O6-BZ pretreated group. A strand bias was observed for GC to TA and AT to TA transversions. Most of the G to A and G to T base substitutions had one or more purines flanking 3' to the mutated deoxyguanosines. There were more deletion mutants with the deletion of exon 1, 4, 6, and 8 in the BZ group than in the control group. These data, characterizing the mutational spectra of ENU in normal human keratinocytes treated in vitro, indicate that GC to AT and AT to GC transition mutations predominate in these cells depleted or not depleted of AGT.

Expression of the O6-alkylguanine-DNA alkyltransferase gene is elevated in human breast tumor cells.

In tumors, resistance to chemotherapeutic drugs that alkylate the O6 position of guanine correlates with the levels of the DNA repair protein, O6-alkylguanine DNA alkyltransferase (AGT). The expression of AGT gene in human breast tumors was evaluated at the level of the single cell, to better understand the distribution of alkylation resistant cells within the tumor. Compared to normal breast ductal cells, the level of AGT expression in the breast tumor cells increased 2-fold. There was no significant association between AGT expression and tumor grade and metastatic malignancy. The up-regulation of AGT was not directly linked to the expression of cyclins D1 and D3, estrogen receptor, p53 and c-erbB-2, genes involved in cell cycle regulation and tumor growth. The elevated expression of AGT in human breast ductal carcinoma cells appeared to be a general characteristic of breast tumors, and suggests that prior treatment with analogs of O6-alkylguanine that inactivate AGT protein, should render the AGT expressing tumor cells sensitive to drugs that alkylate O6-guanine.

Identification and isolation of human epithelial cell colonies that express specific gene products.

A new and simple method for the identification and isolation of human cell clones expressing specific gene products is presented. This technique is analogous to the colony blotting techniques described for prokaryotes. In this technique, human epithelial cells are grown in tissue-culture dishes, and cells from the colonies on the dish are transferred in situ to a high-density cationized quaternary amine-charged nylon membrane. The membrane can then be processed multiple times, using antibody and/or nucleic acid probes, for the identification of those cells producing the desired protein, mRNA or DNA sequence. Once identified, the desired cell colony is isolated for expansion, direct DNA sequencing and/or cell function. We demonstrate the potential of the technique by identifying and isolating colonies of replicating normal human liver hepatocytes producing albumin and keratin.

Differential expression of the O6-alkylguanine-DNA alkyltransferase gene in normal human breast and skin tissue: in situ mapping of cell type-specific expression.

O6-Alkylguanine is a major toxic, mutagenic, and carcinogenic lesion in cellular DNA that is repaired by O6-alkylguanine-DNA alkyltransferase (ATase). The expression of this gene is directly related to the cellular sensitivity of alkylating agents, with levels of this protein varying widely among human organs, tumors, and cell types. To better understand specific cell-type responses to repairing O6-alkylguanine lesions in DNA, we used colorimetric in situ hybridization, with an ATase-specific antisense oligomer probe, to map the cellular distribution of ATase mRNA in tissue sections of normal adult human breast and neonatal foreskin tissues. This is the first report of mapping ATase gene expression directly in normal human breast and skin tissues. Paraffin-embedded tissue sections were hybridized with a digoxigenin-labeled, 39-base antisense ATase oligomer. Hybridization of the probe to cells expressing the ATase gene was visualized after immunodetection with an alkaline phosphatase-conjugated anti-digoxigenin antibody. After color development, we simultaneously identified tissue architecture and cell types and measured the expression of the ATase gene. There was no hybridization-specific color when sections were mock hybridized, hybridized with a sense probe, or treated with RNase. In the breast tissue, 93% of the cells in the loosely connective tissue and 84% of the myoepithelial cells expressed high levels of ATase mRNA. Most of the luminal ductal epithelial cells (61%) were devoid of stain, indicating undetectable levels of ATase mRNA. In skin dermis, 93% of the fibroblasts appeared to express high levels of ATase mRNA. Within the epidermis, approximately 64% of the basal and 65% of the granular epithelial cells expressed ATase mRNA. Expression was undetectable in the epithelial cells of the suprabasal layer of the epidermis. There was very little interindividual variation (< 17%) in the distribution of expression of ATase within the same cell types of different individuals. These data illustrate the differential potential of individual cell types within the organ matrix to repair O6-alkylguanine damage in cellular DNA. This data may provide insights into the understanding of cell type-specific responses to carcinogens.

Cell type-specific expression of human 8-oxo-7,8-dihydroguanosine triphosphatase in normal breast and skin tissues in vivo.

8-Oxo-7,8-dihydroguanosine triphosphate (8-oxo-dGTP) is formed from the oxidation of GTP in the nucleotide pools of cells during normal cellular metabolism and from exogenous sources. 8-Oxo-dGTP is a potent mutagenic substrate for DNA synthesis causing transversion mutations. In human cells this oxidized base is hydrolyzed to 8-oxo-7,8-dihydroguanosine monophosphate by 8-oxo-7,8-dihydroguanosine triphosphatase (8-oxo-dGTPase) to prevent the misincorporation of 8-oxo-dGTP into cellular DNA. In order to better understand specific human tissue and cell type responses to oxidative stress, we used colorimetric in situ hybridization, with an 8-oxo-dGTPase-specific antisense oligomer probe, to map, for the first time, the cellular distribution of 8-oxo-dGTPase mRNA in tissue sections of normal neonatal foreskin and adult human breast tissues. Paraffin embedded tissue sections were hybridized with a digoxigenin-labeled 39 base oligomer, antisense to 8-oxo-dGTPase cDNA. Hybridization of the probe to cells expressing the 8-oxo-dGTPase gene was visualized following immunodetection with an alkaline phosphatase-conjugated anti-digoxigenin antibody. Following color development, we were able to simultaneously identify tissue architecture and cell types with expression of the 8-oxo-dGTPase gene. There was no hybridization-specific color when sections were 'mock' hybridized, hybridized with a sense probe or treated with RNase. In skin dermis, fibroblasts express high levels of 8-oxo-dGTPAse mRNA. Within the epidermis, a gradient of expression was observed, from high to moderate levels in the replicating basal epithelial cells to undetectable in the non-mitotic suprabasal and granular epithelial cells. In the breast tissue, fibroblasts in the loosely connective tissue and myoepithelial cells expressed high levels of 8-oxo-dGTPase mRNA, while expression in the luminal epithelial cells was not detectable. Our data suggest that expression of 8-oxo-dGTP is heterogenous between cell types within an organ and may help to explain cell type-specific responses to oxidative stress, especially in replicating and potentially replicating cells with low levels of this protective protein.

Evaluation of the genotoxicity data on caffeine.

The potential health effects of caffeine have been investigated for over two decades in a variety of model systems including limited human populations. Thus, it is probably one of the most extensively studied natural occurring dietary chemicals. One area which has received a great deal of attention is the potential genotoxic property of caffeine. To better understand whether caffeine itself or in combination with other agents exhibits genotoxic effects, hundreds of research studies published over the past 5 years have been reviewed. These studies have utilized a number of animal, prokaryotic, eukaryotic, and mammalian cell culture model systems. They have investigated the effects of caffeine alone or in combination with other physical and chemical agents on many aspects of cell division, chromosome stability, toxicity, and mutagenicity. A number of effects have been observed. However, they usually appear after very high doses (> 1 mM) of caffeine in combination with genotoxins, and are usually specific to certain cell types and/or cellular parameters. Humans, on the other hand, consume much less caffeine in the diet, with peak serum levels in the micromolar range 10- and 1000-fold higher compared to levels in animal and cell culture models. Thus, it is difficult to implicate caffeine, even at the highest levels of dietary consumption, as a genotoxin to humans.

The establishment and continuous subculturing of normal human adult hepatocytes: expression of differentiated liver functions.

The use of normal adult liver hepatocytes in cell culture for biochemical, toxicological and pharmacological studies has been greatly limited owing to the loss of replicative capacity and differentiated liver function. This is contrary to the ability of the liver to regenerate following injury in vivo. This suggests that liver "stem" or "transitional" hepatocytes exist that upon proper stimulus divide and differentiate into mature hepatocytes. In this study we report the establishment and culture of hepatocytes from normal human adult liver, which: (1) possess replicative capacity sufficient to subpassage 12-15 times (27-37 cumulative population doublings); (2) can be cryopreserved for subsequent use without loss of replicative capacity; and (3) upon differentiation in culture synthesize albumin and keratin 18 and metabolize benzo[a]pyrene. The ability of these cells to divide or express differentiated functions appears to be due to a number of cellular, biochemical and physical characteristics that are present during the primary establishment and subsequent growth phases of the cell cultures. Disassociation of cells from excess liver tissue was best achieved by combining the mechanical action of the Stomacher with very low amounts of proteolytic enzymes and EGTA. The cell lines appeared to grow best when established and subpassaged in an mALPHA medium supplemented with insulin, hydrocortisone, transferrin, epithelial growth factor and fetal bovine serum (prescreened for human hepatocyte cell growth). The seeding density and cell-cell contact in culture appeared to be important for both cell division and expression of liver function. When cells were seeded at a low density and subpassaged before confluency, the cells continued to divide. Albumin and keratin 18 synthesis occurred primarily in tightly packed cell clusters. When cells were seeded at a high density, near confluency, albumin and keratin 18 synthesis occurred uniformly in all of the cells of the culture and the culture metabolized benzo[a]pyrene to water-soluble metabolites, which covalently bound to cellular DNA. This appearance of liver functions was consistent with the "transition" of hepatocytes to a terminally differentiated state. Nonhepatic markers, i.e., alpha-fetoprotein, factor VIII and gamma-glutamyl transpeptidase activity were not expressed in cells cultured at either low or high density. Thus, the data presented here indicate that normal human adult liver hepatocytes, once established in culture, can be subpassaged to a high number of population doublings, cryopreserved for later use, and modulated to express differentiated liver functions.