The effect of ethanol and nicotine on ER stress in human placental villous explants.

Pregnant mothers continue smoking and drinking during pregnancy. To clarify the mechanisms of nicotine and ethanol toxicity during development, we have examined their effects on endoplasmic reticulum (ER) stress in human first trimester and term placental explants. First trimester and term human placental explants were treated with ethanol (2 ‰) or nicotine (15 µM), or their combination. The ER stress markers glucose regulated protein 78 (GRP78/BiP) and inositol requiring enzyme 1 α (IRE1α) were analyzed by immunoblotting. A statistically significant increase (p < 0.05) of GRP78/BiP by nicotine was noted in first trimester placental explants at 48 h, and in term placental explants at 24 h. Ethanol did not change protein expression of GRP78/BiP in either first trimester or term placental explants. IRE1α increased, although not statistically significantly, by all treatments in both first trimester and term placental explants. Thus, regardless of the known structural and functional differences in early and late placenta, both responded very similarly to the toxic compounds studied. These data support our earlier results in BeWo cells (Repo et al., 2014) implicating that nicotine induces ER stress in human placenta and may interfere with placental functions potentially disrupting fetal growth and development.

Criteria and challenges of the human placental perfusion ­ Data from a large series of perfusions.

Perfusion of human placental cotyledon has been used extensively to study transplacental transfer of endogenous and exogenous compounds. However, many challenges in the use of the method exist, including availability of placentas and complexity of the method itself. In Kuopio, Finland we have carried out human placental perfusions since 2005 using the same method with data now from over one hundred perfusions. This has allowed us to study whether the way of delivery, placental weight, and/or the length of pregnancy affect the two major criteria of a successful perfusion: volume loss (leak) from fetal to maternal circulation, and transplacental transfer of the reference compound antipyrine. The only statistically significant result was the reduction of the fetomaternal ratio of antipyrine by the placental age over 40 weeks (p=0.0004). The success criteria were not affected by the weight of the placenta or the way of delivery. There was no effect by the antipyrine concentration on antipyrine transfer. In vitro incubation with different concentrations of study compounds and different tubing materials could offer an easy way to study potentially reduced recovery due to binding to perfusion system.

Benzo(a)pyrene increases phosphorylation of p53 at serine 392 in relation to p53 induction and cell death in MCF-7 cells.

Although benzo(a)pyrene (BP) induces apoptosis in vitro in murine Hepa1c1c7 cells and in vivo indications of apoptosis in rat lung exist, related cellular mechanisms in human cells are not known. p53 protein participates in several apoptotic processes. We found that BP induces cell death in human MCF-7 breast adenocarcinoma cells at 48 and 72h but not in human A549 lung carcinoma cells. BP did not induce measurable caspase-3-like protease activity or internucleosomal DNA fragmentation in either cell types. However, procaspase-7 cleavage in MCF-7 cells by BP-treatment indicates activation of caspase-7 meaning that apoptosis is most likely involved in BP-induced MCF-7 cell death. BP-7,8-dihydrodiol-9,10-epoxide (BPDE)-DNA adducts and level of p53 protein increased dose-dependently, but more extensively in MCF-7 cells. Phosphorylation of p53 protein at serines 15, 20, 46 and 392 increased in MCF-7 cells. Increase in phosphorylation at serine 392 was clear already at 24h by 1 microM concentration of BP. Increase of phosphorylation at other sites occurred only with higher concentrations or at later time points in relation to the increase of p53 protein. These results suggest that serine 392 phosphorylation is the first stabilizing event of p53 associated with BP exposure and subsequent cell death in MCF-7 cells.

Ethics in studies on children and environmental health.

Children, because of age-related reasons, are a vulnerable population, and protecting their health is a social, scientific and emotional priority. The increased susceptibility of children and fetuses to environmental (including genotoxic) agents has been widely discussed by the scientific community. Children may experience different levels of chemical exposure than adults, and their sensitivity to chemical toxicities may be increased or decreased in comparison with adults. Such considerations also apply to unborn (fetal exposure) and newborn (neonatal exposure) children. Therefore, research on children is necessary in both clinical and environmental fields, to provide age-specific relevant data regarding the efficacy and safety of medical treatments, and regarding the assessment of risk from unintended environmental exposure. In this context, the stakeholders are many, including children and their parents, physicians and public health researchers, and the society as a whole, with its ethical, regulatory, administrative and political components. The important ethical issues are information of participants and consent to participate. Follow-up and protection of data (samples and information derived from samples) should be discussed in the context of biobanks, where children obtain individual rights when they become adults. It is important to realise that there are highly variable practices within European countries, which may have, in the past, led to differences in practical aspects of research in children. A number of recommendations are provided for research with children and environmental health. Environmental research with children should be scientifically justified, with sound research questions and valid study protocols of sufficient statistical power, ensuring the autonomy of the child and his/her family at the time of the study and later in life, if data and samples are used for follow-up studies. When children are enrolled, we recommend a consent dyad, including (1) parental (or legal guardian) informed consent and (2) the child's assent and/or informed consent from older minors. For evaluation of the studies including children, a paediatrician should always be involved in the research ethics committee.

Is there a role for PCR-SSCP among the methods for missense mutation detection of TP53 gene?

Mutation analysis methods have increased in variety during the past years. High-throughput microarray methods have especially increased in popularity. However, new methods require reference points, and not all of the methods are equal in sensitivity and specificity. Furthermore, the detection of unknown missense mutations, such as unknown TP53 mutations in human tumors, for clinical purposes requires great accuracy, which may be difficult to acquire with the current high-throughput methods. For these reasons, the classical methods, such as PCR-manual sequencing and PCR-SSCP, are still valuable and necessary.

Effect of glutamate and extracellular calcium on uptake of inorganic lead (Pb2+) in immortalized mouse hypothalamic GT1-7 neuronal cells.

We have previously shown that although glutamate alone has no effects on viability of mouse hypothalamic GT1-7 cells, it clearly enhances Pb2+-induced cytotoxicity. It is likely that Pb2+ must enter cells to exert most of its toxic effects. Pb2+ is known to substitute for Ca2+ in many cellular processes. Therefore, we studied the uptake mechanisms of Pb2+ into GT1-7 neuronal cells with a special focus on the role of extracellular calcium (Ca2+), voltage-sensitive calcium channels (VSCCs) and glutamate. Basal uptake of Pb2+ (1 microM or 10 microM), i.e. without any external stimulus, clearly increased in nominally Ca2+-free buffer and was partially abolished by 13 mM Ca2+ when compared to uptake in the presence of a physiological concentration of extracellular Ca2+ (1.3 mM). Depolarization by 25 mM K+, or antagonists of VSCCs, verapamil (10 microM) or flunarizine (10 microM) had no clear effect on basal Pb2+ uptake. Glutamate (1 mM) increased Pb2+ uptake, but only when cells were treated with 1 microM Pb2+ in the presence of 1.3 mM Ca2+. Our data suggest that Pb2+ competes for the same cellular uptake pathways with Ca2+, although not via VSCCs. In addition, enhancement of Pb2+-induced neurotoxicity by glutamate may be due to increased neuronal uptake of Pb2+.

p53 and K-ras mutations in lung cancers from former and never-smoking women.

Somatic p53 mutations are common in lung cancer. Active cigarette smoking is positively correlated with the total frequency of p53 mutations and G:C to T:A transversions on the nontranscribed (DNA coding) strand. Mutational hotspots within the p53 gene, e.g., codon 157, have been identified for tobacco-related lung cancer, whereas these same mutations are found rarely in other cancers. Such data implicate specific p53 mutations as molecular markers of smoking. Because limited data exist concerning the p53 mutation frequency and spectra in ex-smokers and nonsmokers, we have analyzed p53 and K-ras mutations in 126 lung cancers from a population-based case-control study of nonsmoking (n = 117) or ex-smoking (n = 9) women from Missouri with quantitative assessments of exposure to environmental tobacco smoke. Mutations in the p53 gene were found in lung cancers from lifetime nonsmokers (19%) and ex-smokers (67%; odds ratio, 9.08; 95% confidence interval, 2.06-39.98). All deletions were found in tumors from patients who were either ex-smokers or nonsmokers exposed to passive smoking. The G:C to A:T transitions (11 of 28; 39%) were the most frequent p53 mutations found and clustered in tumors from lifetime nonsmokers without passive smoke exposure. The incidence of K-ras codon 12 or 13 mutations was 11% (14 of 115 analyzed) with no difference between long-term ex-smokers and nonsmokers. These and other results indicate that p53 mutations occur more commonly in smokers and ex-smokers than in never-smokers. Such comparisons provide additional evidence of genetic damage caused by tobacco smoke during lung carcinogenesis.

Single-Strand Conformation Polymorphism Analysis of Mutations in Exons 4-8 of the TP53 Gene.

The TP53 tumor suppressor gene coding for a nuclear phosphoprotein involved in cellular stress responses is the most frequently mutated gene in human cancers described so far (1-4). Mutations are found throughout the gene but most frequently within the highly conserved middle region (exons 5-8) that encodes for the DNA-binding central region of the gene critical for the major function of TP53 protein as a transcriptional activator (5). The mutation spectrum of the TP53 gene varies from one tumor type to another with typical hot-spot codons for mutations (2,6). For instance, codons 157, 248, and 273 are frequently mutated in cigarette smoking-associated lung cancer, whereas mutations in codon 175 are rare. This codon, on the other hand, is often mutated in breast and colon cancer. In some cases, typical mutations can be linked with environmental exposures, such as CC→TT double mutation with UV radiation (7) and codon 249 AGG→AGT mutation with aflatoxin B1 and hepatitis B virus (8). These findings, in connection with the fact that one of the main functions of TP53 protein is putatively the protection of the genome (9,10) implicate the mutations of the TP53 gene in environmentally induced carcino-genesis in humans and the possible use of TP53-related markers in molecular epidemiology (11-13).

Environmental tobacco smoke, genetic susceptibility, and risk of lung cancer in never-smoking women.

BACKGROUND: Exposure to environmental tobacco smoke (ETS) is considered to be a major lung cancer risk factor for never smokers. We investigated the hypothesis that never-smoking women who are exposed to ETS and develop lung cancer are a genetically susceptible population. METHODS: Archival tumor tissues were analyzed from 106 never-smoking women enrolled in a case-control study of ETS (and other personal and environmental factors) and lung cancer risk. We analyzed germline polymorphisms in genes that have been associated with cancer susceptibility and whose products activate (cytochrome P450 1A1 [CYP1A1]) and detoxify (glutathione S-transferases M1 [GSTM1] and T1 [GSTT1]) chemical carcinogens found in tobacco smoke. RESULTS: When compared with never smokers who had no ETS exposure and developed lung cancer (n = 55), never smokers with exposure to ETS who developed lung cancer (n = 51) were more likely to be deficient in GSTM1 activity (i.e., were GSTM1 null) because of a genetic polymorphism in the GSTM1 gene (odds ratio = 2.6; 95% confidence interval = 1.1-6.1). A statistically significant rising trend in risk occurred with increasing ETS exposure (two-sided P =. 02), reaching a more than sixfold excess risk in those exposed to 55 pack-years of ETS (ETS pack-year = ETS produced by an active smoker, within a confined space such as a room, who smokes one pack of cigarettes a day for a year). No evidence was found of associations between GSTT1 deficiency or the CYP1A1 valine variant and lung cancer risk due to ETS exposure. CONCLUSIONS: A common genetic polymorphism divides the population of never smokers into two groups of approximately equal size, one (homozygous carriers of the GSTM1 null allele) that has a statistically significant greater risk of lung cancer from ETS than the other (heterozygous or homozygous carriers of the wild-type GSTM1 allele).

Molecular epidemiology of human cancer risk: gene-environment interactions and p53 mutation spectrum in human lung cancer.

The p53 tumour suppressor gene is at the crossroads of a network of cellular pathways including cell cycle checkpoints, DNA repair, chromosomal segregation, and apoptosis. These pathways have evolved to maintain the stability of the genome during cellular stress from DNA damage, hypoxia, and activated oncogenes. The high frequency of p53 mutations in human cancer is a reflection of the importance of p53 involvement in this network of pathways during human carcinogenesis. An electronic database containing p53 mutations from more than 9000 cancers (http:/(/)www.iarc.fr/p53/homepage.html) can be used to generate hypotheses for further clinical, epidemiological, and laboratory investigations. For example, one can hypothesize that (a) p53 mutations vary in their pathobiological significance; (b) cellular content influences the selection of p53 mutations in clonally derived cancers; (c) the location and type of mutation within the p53 gene provide clues to functional domains in the gene product; and (d) the p53 mutation spectrum can be a molecular link between aetiological agents and human cancer. This review will focus on the role of p53 and cancer susceptibility genes in the molecular pathogenesis and epidemiology of human lung cancer.

Placental transfer of sulindac, sulindac sulfide, and indomethacin in a human placental perfusion model.

OBJECTIVE: Evaluation of the transplacental transfer and placental metabolism of sulindac, its active sulfide metabolite, and indomethacin, drugs used as tocolytic agents, in dual recirculating human placental perfusion. STUDY DESIGN: Term placentas were obtained with maternal consent immediately after delivery. Drugs were added to the maternal reservoir, together with antipyrine as a reference compound, and disappearance from the maternal circulation and appearance in the fetal circulation were followed up for 2 hours in 4 experiments for each compound. Drug concentrations were analyzed by high-performance liquid chromatography. RESULTS: The fetal/maternal concentration ratios after 2-hour perfusions were 0. 34 +/- 0.19 (mean +/- SD, sulindac), 0.54 +/- 0.17 (sulfide), and 0. 45 +/- 0.16 (indomethacin), and the fetal-maternal transfer percentages at 2 hours were 11.6 +/- 5.9 (sulindac), 18.2 +/- 5.2 (sulfide), and 15.3 +/- 4.5 (indomethacin). No metabolism of sulindac or indomethacin was detected. CONCLUSION: Sulindac sulfide, formed through hepatic metabolism, reaches the fetus in higher concentrations than does sulindac or indomethacin. Neither sulindac nor indomethacin is metabolized by the human placenta.

Infrequent p53 mutations in arsenic-related skin lesions.

Oral arsenic exposure increases the risk for a variety of benign and malignant skin lesions, but the molecular mechanism of the carcinogenic effect is poorly understood. Arsenic-related squamous cell carcinomas of the skin can develop either de novo or progress from Bowen's disease lesions. Arsenic-related basal cell carcinomas develop usually in non-sun-exposed areas and are multiple. Because p53 tumor suppressor protein is a protective cellular molecule against environmental carcinogens and mutations in the p53 gene are frequent in nonmelanoma skin cancers, we studied p53 in 23 premalignant or malignant skin lesions from seven patients with a history of arsenic medication. The eighth patient studied (with six lesions) had a long standing exposure to UV radiation. Accumulation of the p53 protein was detected (with a monoclonal DO-7 antibody) in 78% of the lesions from cases with arsenic exposure. Two of the six (30%) arsenic-related premalignant lesions and in addition one UV related carcinoma in situ lesion were clearly and repeatedly positive when p53 exons 5 to 8 were screened by a nonradioactive single-strand conformation polymorphism (SSCP) analysis. Only one of the arsenic-related lesions was confirmed by sequencing to have a mutation (a CC to TT double transition). No indications of mutations were found among the 18 basal cell carcinoma or two squamous cell carcinoma lesions studied. Our results suggest that the frequent accumulation of p53 protein in arsenic-related skin lesions is not due to p53 mutations. which may not be a prerequisite in the development of arsenic-induced skin cancers.

Single-strand conformation polymorphism analysis to detect p53 mutations: characterization and development of controls.

Single-strand conformation polymorphism (SSCP) analysis is widely used to prescreen mutations in p53 gene. However, standardization of SSCP to detect p53 mutations has rarely been pursued so far. We have developed complete conditions for a temperature-controlled nonradioactive SSCP for mutation detection in amplified p53 exons 4-8, where mutations frequently occur in human tumors. Easily obtainable and clearly distinguishable positive controls were developed by replacing the regular 5' primers in amplification with primers that include one to three mutated sites. Careful purification of the amplified products by gel electrophoresis appeared to be essential. The efficiency of the method was studied by using previously sequenced samples with p53 mutations and the various positive controls. The use of two temperatures (exon 4: 4 degrees C and 15 degrees C; other exons: 4 degrees C and 20 degrees C) in combination with other optimized conditions resulted in 98% efficiency in mutation detection, which was considered sufficient for routine screening.

Mutations of p53 and ras genes in radon-associated lung cancer from uranium miners.

Radon increases the risk of lung cancer in smoking and non-smoking underground miners. To investigate the mutational spectrum associated with exposure to high levels of radon, we sequenced exons 5-9 of the p53 tumour suppressor gene and codons 12-13 of the Ki-ras protooncogene in 19 lung cancers from uranium miners exposed to radon and tobacco smoke. Mutations were not found in Ki-ras, but 9 p53 mutations, including 2 deletions, were found in 7 patients by direct DNA sequencing after polymerase chain reaction amplification of DNA from formalin-fixed, paraffin-embedded tissue. In tumours from 5 patients, the mutation produced an aminoacid change and an increased nuclear content of p53 protein. The tumours with either a stop codon or frame-shift deletion in the p53 gene were negative by immunohistochemistry. None of the mutations were G:C to T:A transversions in the coding strand of the p53 gene, which are the most frequent base substitutions associated with tobacco smoking, and none were found at the hotspot codons described in lung cancer. The observed differences from the usual lung cancer mutational spectrum may reflect the genotoxic effects of radon.