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Background: DNA damage caused by exposure to metal mixtures and the potential modulating role of genes involved in DNA repair and the antioxidant response have not been evaluated in newborns., Aim: The aim was to evaluate the association between prenatal exposure to metal mixtures and DNA repair capacity (DRC) in newborns from the Metropolitan Area of Mexico City (MAMC), a heavily polluted area, and the impact of variants in genes involved in DNA repair and the antioxidant response on this association., Methods: We analyzed cord blood samples obtained at delivery from 125 healthy newborns from the MAMC. Twenty-four elements were determined by inductively coupled plasma mass spectrometry (ICP‒MS), but only 12 (Cu, I, Se, Zn, As, Ba, Cs, Mn, Sb, Sr, Pb, and Ti) were quantified in most samples. DRC was assessed by the challenge-comet assay, and OGG1, PARP1, and NFE2L2 genotyping was performed with TaqMan probes. Metal mixtures were identified and analyzed using principal component analysis (PCA) and weighted quantile sum (WQS) regression. Independent adjusted linear regression models were used to evaluate the associations., Results: A null DRC was observed in 46% of newborns. The metals with the highest concentrations were Mn, Sr, Ti, and Pb. Essential elements showed normal levels. Only the mixture characterized by increased As, Cs, Cu, Se, and Zn levels was inversely associated with DRC. As was the principal contributor (37.8%) in the negative direction in the DRC followed by Ba and Sb, according to the WQS regression. Newborns carrying of the derived (G) allele of the PARP1 rs1136410 variant showed decreased DRC by exposure to some potentially toxic metals (PTMs) (As, Cs, and Ba)., Conclusion: Prenatal exposure to metal mixtures negatively affected DRC in newborns, and the PARP1 rs1136410 variant had a modulating role in this association., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Pathways that regulate protein homeostasis (proteostasis) in cells range from mRNA processing to protein degradation; perturbations in regulatory mechanisms of these pathways can lead to oncogenic cellular processes. Protein synthesis modulation failures are common phenomena in cancer cells, wherein specific conditions that promote the translation of protein factors promoting carcinogenesis are present. These specific conditions may be favored by metabolic lipid alterations like those found in metabolic syndrome and obesity. Protein translation modifications have been described in obesity, favoring the translation of protein targets that benefit lipid accumulation; a determining factor is the activity of the cap-binding eukaryotic translation initiation factor 4E (eIF4E), a crosstalk in protein translation and lipogenesis. Besides, alterations of protein translation initiation steps are critical participants for the development of both pathogenic conditions, cancer, and obesity. This chapter is focused on the regulation of recognition and processing of carcinogenic-mRNA and the connections among lipid metabolism and cell signaling pathways that promote oncogenesis, tumoral microenvironment generation and potentially the development of chemoresistance. We performed an in-depth analysis of events, such as those occurring in obesity and dyslipidemias, that may influence protein translation, driving the recognition of certain mRNAs and favoring cancer development and chemoresistance., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Methyl parathion (Me-Pa) is an extremely toxic organophosphorus pesticide still used in developing countries. It has been associated with decreased sperm function and fertility and with oxidative and DNA damage. The blood-testis barrier (BTB) is a structure formed by tight junction (TJ) proteins in Sertoli cells and has a critical role in spermatogenesis. We assessed the effect of repeated doses of Me-Pa (3-12 mg/kg/day for 5 days, i.p.) on sperm quality, lipid oxidation, DNA integrity, and BTB permeability in adult male mice and explored oxidation as a mechanism of toxicity. Me-Pa caused dose-dependent effects on sperm quality, lipoperoxidation, and DNA integrity. Testis histology results showed the disruption of spermatogenesis progression and atrophy of seminiferous tubules. The pesticide opened the BTB, as evidenced by the presence of a biotin tracer in the adluminal compartment of the seminiferous tubules. This effect was not observed after 45 days of exposure when a spermatogenic cycle had completed. The coadministration of the antioxidant α-tocopherol (50 mg/kg/day for 5 days, oral) prevented the effects of Me-Pa on sperm quality, DNA and the BTB, indicating the importance of oxidative stress in the damage generated by Me-Pa. As evidenced by immunochemistry, no changes were found in the localization of the TJ proteins of the BTB, although oxidation (carbonylation) of total proteins in testis homogenates was detected. Our results show that Me-Pa disturbs the BTB and that oxidation is involved in the observed toxic effects on sperm cells., Competing Interests: Declaration of Competing Interest The authors have no competing interests to declare., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Di(2-ethylhexyl) phthalate (DEHP) is a widely used plasticizer that is metabolized to mono(2-ethylhexyl) phthalate (MEHP). Inhalation is an important exposure route for both phthalates, and their effects on lungs include inflammation, alteration of postnatal maturation (alveolarization), enlarged airspaces and cell differentiation changes, suggesting that alveolar epithelial cells-2 (AEC) are targets of phthalates. This study evaluated the cell progression, epithelial and mesenchymal markers, including surfactant secretion in A549 cells (AEC) that were exposed to DEHP (1-100 μM) or MEHP (1-50 μM) for 24-72 h. The results showed an increased cell proliferation at all concentrations of each phthalate at 24 and 48 h. Cell migration showed a concentration-dependent increase at 24 and 48 h of exposure to either phthalate and enlarged structures were seen. Decreased levels of both surfactants (SP-B/SP-C) were observed after the exposure to either phthalate at 48 h, and of SP-C positive cells exposed to MEHP, suggesting a loss of the epithelial phenotype. While a decrease in the epithelial marker E-cadherin and an increase in the mesenchymal marker fibronectin were observed following exposure to either phthalate. Our results showed that DEHP and MEHP altered the structure and migration of A549 cells and promoted the loss of the epithelial phenotype., (Copyright © 2018 Elsevier B.V. All rights reserved.)

DNA methylation in DNA repair genes participates in the DNA damage regulation. Particulate matter (PM), which has metals and polycyclic aromatic hydrocarbons (PAHs) adsorbed, among others has been linked to adverse health outcomes and may modify DNA methylation. To evaluate PM exposure impact on repetitive elements and gene-specific DNA methylation and DNA damage, we conducted a cross-sectional study in 150 schoolchildren (7-10 years old) from an urbanized, industrial area of the metropolitan area of Mexico City (MAMC), which frequently exhibits PM concentrations above safety standards. Methylation (5mC) of long interspersed nuclear element-1 (LINE1) and DNA repair gene (OGG1, APEX, and PARP1) was assessed by pyrosequencing in peripheral mononuclear cells, DNA damage by comet assay and DNA oxidation by 8-OHdG content. PAH and metal contents in PM 10 (≤10μm aerodynamic diameter) were determined by HPLC-MS and ICP-AES, respectively. Multiple regression analysis between DNA methylation, DNA damage, and PM 10 exposure showed that PM 10 was significantly associated with oxidative DNA damage; a 1% increase in 5mC at all CpG sites in PARP1 promoter was associated with a 35% increase in 8-OHdG, while a 1% increase at 1, 2, and 3 CpG sites resulted in 38, 9, and 56% increments, respectively. An increase of 10pg/m 3 in benzo[b]fluoranthene content of PM 10 was associated with a 6% increase in LINE1 methylation. Acenaphthene, indene [1,2,3-cd] pyrene, and pyrene concentrations correlated with higher dinucleotide methylation in OGG1, APEX and PARP1 genes, respectively. Vanadium concentration correlated with increased methylation at selected APEX and PARP1 CpG sites. DNA repair gene methylation was significantly correlated with DNA damage and with specific PM 10 -associated PAHs and Vanadium. Data suggest that exposure to PM and its components are associated with differences in DNA methylation of repair genes in children, which may contribute to DNA damage., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Stress is a complex event that induces disturbances to physiological and psychological homeostasis, and it may have a detrimental impact on certain brain and physiological functions. In the last years, a dual role of the stress effect has been studied in order to elucidate the molecular mechanism by which can induce physiological symptoms after psychological stress exposition and vice versa. In this sense, inflammation has been proposed as an important starring. And in the same line, the inflammasome complex has emerged to give responses because of its role of stress sensor. The implication of the same complex, NLRP3 inflammasome, in different diseases such as cardiovascular, neurodegenerative, psychiatric, and metabolic diseases opens a door to develop new therapeutic perspectives., (© 2017 Elsevier Inc. All rights reserved.)

Currently there are no biological markers to indicate which individuals with chronic indeterminate period of Chagas disease develop heart disease and who will remain all his life in this phase. The aim of this survey was to determine if Trypanosoma cruzi burden is related to the presence of heart disease in patients with chronic Chagas disease. 200 patients who had not been treated, 100 with cardiopathy and 100 without, groups A and B respectively, were submitted to clinical study and electrocardiogram, Echo-Doppler was performed for group A in which all important known causes of cardiopathy were discarded. In both groups xenodiagnosis, conventional PCR and quantitative PCR were undertaken. The 100 cardiopaths had 133 electrocardiographic alterations most of them in grade II of the New York Heart Association classification. 98 cardiopaths were classified in grade I by Echo-Doppler and only 2 cases were in grade III due to low ejection fraction. The difference in average parasitemia in patients of group A and B was not significant and no statistically differences were observed between average parasitemia of cardiopaths grade II versus grade I of NYHA. This results allow to characterize same clinical, electrocardiographical and parasitological features in chagasic cardiopaths of Chile., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The Community-Embedded Reproductive Health Care for Adolescents (CERCA) Project was implemented in Bolivia, Ecuador and Nicaragua (2011-2014) to test the effectiveness of interventions preventing teenage pregnancies. As the outcome evaluation showed limited impact, a post-hoc process evaluation was carried out to determine if and how CERCA's design, implementation, monitoring and evaluation affected the results. We did a document analysis and conducted 18 in-depth interviews and 21 focus group discussions with stakeholders and beneficiaries. Transcripts were analyzed using directed content analysis. Data showed that CERCA sensitized stakeholders and encouraged the discussion on this sensitive issue. In terms of design, a strong point was the participatory approach; a weak point was that the detailed situation analysis was completed too late. In terms of implementation, a strong point was that multifaceted activities were implemented; a weak point was that the activities were not pilot tested for feasibility/acceptability and evolved substantially throughout the Project. In terms of monitoring, strong points were that regular monitoring kept the Project on track administratively/financially; a weak point was that monitoring indicators did not change as the intervention package changed. In terms of evaluation, weak points were the substantial attrition rate and narrow focus on adolescents. This study provides recommendations for future projects., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Immunoglobulin light-chain amyloidosis (AL) and multiple myeloma (MM) are 2 distinct monoclonal gammopathies that involve the same cellular compartment: clonal plasma cells (PCs). Despite the fact that knowledge about MM PC biology has significantly increased in the last decade, the same does not apply for AL. Here, we used an integrative phenotypic, molecular, and genomic approach to study clonal PCs from 24 newly diagnosed patients with AL. Through principal-component-analysis, we demonstrated highly overlapping phenotypic profiles between AL and both monoclonal gammopathy of undetermined significance and MM PCs. However, in contrast to MM, highly purified fluorescence-activated cell-sorted clonal PCs from AL (n = 9) showed almost normal transcriptome, with only 38 deregulated genes vs normal PCs; these included a few tumor-suppressor (CDH1, RCAN) and proapoptotic (GLIPR1, FAS) genes. Notwithstanding, clonal PCs in AL (n = 11) were genomically unstable, with a median of 9 copy number alterations (CNAs) per case, many of such CNAs being similar to those found in MM. Whole-exome sequencing (WES) performed in 5 AL patients revealed a median of 15 nonrecurrent mutations per case. Altogether, our results show that in the absence of a unifying mutation by WES, clonal PCs in AL display phenotypic and CNA profiles similar to MM, but their transcriptome is remarkably similar to that of normal PCs., (© 2016 by The American Society of Hematology.)

Introduction: The PIBHE study, promoted by the Spanish Liver and Kidney Association and the Dialysis Virus Group of the Spanish Society of Nephrology, is the first study to determine the status of haemodialysis patients with chronic HBV infection and the immunisation against the vaccine., Method: The study has a national multicentre, observational, cross-sectional design and was carried out between January 2013 and 2014. A data collection folder was sent to all the nephrology departments and outpatient haemodialysis units in Spain, to be completed based on patient medical files after informed consent. The data were recorded in a central database., Results: A total of 215 centres participated (15,645 patients), with an HBV prevalence of 1.03%. HCV or HIV was present in 7.2% of the HBV(+) patients. Viral load was below 2,000 IU/ml in 80%. GOT and GPT levels were 19.1±10.1 and 15.9±9.6 IU/ml, respectively. Liver biopsy was performed in 7.1%. Antiviral treatment was prescribed in 30% and suspended in 12.5%: entecavir (13.3%), lamivudine (10%), adefovir and tenofovir (6.7%), and interferon (3.3%). A total of 34.5% were candidates for renal transplantation and 6.9% had not been evaluated; 64.3% were followed up by a gastroenterologist; 27.2% of HBV(-) patients without immunisation had not been vaccinated. Fourteen different immunisation schedules had been used, with an immunisation rate of 58.8%. Mean anti-HBs stood at 165.7±297.8mIU/ml. A total of 72.7% of patients had received a vaccination course; 26.4%, 2 cycles; 1.0%, 3 cycles; and 11.6%, a booster dose. A total of 28.3% had a poor response (anti-HBs 10-99mIU/ml); 22.4%, an optimal response (anti-HBs 100-999mIU/ml); and 7.9%, an excellent response (anti-HBs ≥ 1,000mIU/ml). Age was significantly associated with response to vaccination; the mean age of nonresponders was significantly higher than patients who had a response of any kind (P<.05). The highest probability of an immune response was achieved with 4 doses of 40 mcg of adjuvanted vaccine (OR: 7.3; 95% CI 3.4 to 15.7), for the same age and number of cycles and boosters. Age, adjuvanted vaccine, dose and vaccination schedule influenced the immune response and the anti-HBs titres reached (P<.05)., Conclusion: The prevalence of chronic HBV infection in haemodialysis in Spain is low and so are the rates of immunisation against the virus. The vaccination schedules used are very diverse and have been observed to correlate with the immune response. It would therefore be necessary to establish a protocol for the most effective vaccination schedule to increase immunisation in these patients., (Copyright © 2015 Sociedad Española de Nefrología. Published by Elsevier España, S.L.U. All rights reserved.)

Leptin is a hormone that regulates energy homeostasis, inflammation, hematopoiesis and immune response, among other functions (Houseknecht et al., 1998; Zhang et al., 1995; Paz-Filho et al., 2010). To obtain its crystallographic structure, it was necessary to substitute a tryptophan for a glutamic acid at position 100, thus creating a mutant leptin that has been reported to have biological activity comparable to the activity of the wild type but that crystallizes more readily. Here, we report a comparative study of the conformational space of WT and W100E leptin using molecular dynamics simulations performed at 300, 400, and 500 K. We detected differences between the interactions of the two proteins with local and distal effects, resulting in changes in the conformation, accessible surface area, compactness, electrostatic potential and dynamic behavior. Additionally, the series of unfolding events that occur when leptin is subjected to high temperature differs for the two constructs. We observed that both proteins are mostly unstructured after 20 ns of MD simulation at 500 K. However, WT leptin maintains a significant amount of secondary structure in helix α2, while the most stable region of W100E leptin is helix α3. Furthermore, we found that the region between residues 25 and 42 might adopt interconverting secondary structures ranging from α-helices and random coils to β-strand structures. Thus, this region can be considered an intrinsically disordered region. This atomistic description supports our understanding of leptin signaling and consequently might facilitate the use of leptin in treatments for the pathophysiologies in which it is implicated., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Surface plasmon resonance (SPR)-based biosensors are established tools for measuring biomolecular interactions between unlabeled analytes in real time, and are thus an ideal method to evaluate G protein-coupled receptor (GPCR) binding interactions. Using as a vehicle lentiviral particles bearing the chemokine receptor CXCR4 in its native plasma membrane context, SPR analysis can be performed using the particles as specific receptors to monitor the CXCR4 interaction with its ligand, CXCL12. The method shows linear correlation in the 5-40 nM range, with low intra- and inter-assay variation, a relative standard deviation <10%, chip-to-chip variation <12%, with stability of the sensor response for more than 150 measurements in the same chip over a four-week period. Our objective was to develop a method for rapid detection and quantification of analytes such as CXCL12 in biological samples, with no need for pretreatment. As a proof of concept, we tested for CXCL12 in urine samples from rheumatoid arthritis patients, who have elevated levels of this chemokine in plasma and synovial fluid. The biosensor method allowed sensitive, reproducible CXCL12 detection in the physiological range, suggesting its value for the diagnosis of autoimmune disorders., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants presenting a public health risk, particularly to children, a vulnerable population. PAHs have genotoxic and carcinogenic properties, which depend on their metabolism. Many enzymes involved in PAH metabolism, including CYP1A1, CYP1B1, GSTM and GSTT are polymorphic, which may modulate the activation/deactivation of these compounds. We evaluated PAH exposure and DNA damage in children living in the vicinity of the main petrochemical complex located in the Gulf of Mexico, and explored the modulation by genetic polymorphisms of PAH excretion and related DNA damage. The participants (n=82) were children aged 6-10y attending schools near the industrial area. Urinary 1-hydroxypyrene (1-OHP; a biomarker of PAH exposure) was determined by reverse-phase-HPLC; DNA damage by the comet assay (Olive Tail Moment (OTM) parameter); CYP1A1*2C and CYP1B1*3 polymorphisms by real time-PCR; and GSTM1*0 and GSTT1*0 by multiplex PCR. The median value of 1-OHP was 0.37μmol/mol creatinine; 59% of children had higher 1-OHP concentrations than those reported in environmentally exposed adults (0.24μmol/mol creatinine). A stratified analysis showed increased DNA damage in children with 1-OHP concentrations greater than the median value. We observed higher 1-OHP concentrations in children with CYP1A1*2C or GSTM1*0 polymorphisms, and a positive influence of CYP1A1*2C on OTM values in children with the highest PAH exposure. The data indicate that children living in the surroundings of petrochemical industrial areas are exposed to high PAH levels, contributing to DNA damage and suggesting an increased health risk; furthermore, data suggest that polymorphisms affecting activation enzymes may modulate PAH metabolism and toxicity., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: The involvement of iron in anthracycline cardiotoxicity is supported by extensive experimental data, and by the preventive efficacy of dexrazoxane, an iron chelator. However, no clinical evidence of anthracycline-induced cardiac iron accumulation is available and the influence of previous iron overload or of genetic factors in human-induced heart disease is largely unknown. Our aim was to test the hypothesis that anthracyclines increase iron heart concentration and that HFE genotype modulates this iron deposit., Methods: We retrospectively evaluated cardiac events, cardiac iron and HFE genotype in 97 consecutive necropsies from patients with solid and hematological neoplasms. Heart and liver iron concentration was determined by atomic absorption spectroscopy. HFE gene mutations (C282Y and H63D) linked to hereditary hemochromatosis were analyzed by Fluorescence Resonance Energy Transfer (FRET) genotyping., Results: Heart iron concentration was increased in cases treated with a cumulative doxorubicin dose greater than 200mg/m(2) (490 vs 240 μg/g; p=0.01), independently of liver iron load or transfusion history. HFE mutated haplotypes 282C/63D (p=0.049) and 282Y/63H (p=0.027) were associated to higher cardiac iron deposits. The haplotype C282Y-Y/H63D-H interacted with anthracyclines for increasing cardiac iron load. In a multivariate linear regression analysis both HFE genotypes and anthracyclines contributed to heart iron concentration (R(2)=0.284)., Conclusions: Our data support the occurrence of an HFE-modulated heart iron accumulation in individuals treated with anthracyclines, independently of systemic iron load. If prospectively confirmed, iron-related parameters might be useful as predictive factors for anthracycline cardiotoxicity., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Paraoxonase (PON1) enzyme is associated with high-density lipoproteins (HDL) that prevents low-density lipoprotein (LDL) oxidation. PON1Q192R polymorphism is associated with a risk of coronary heart disease and low HDL levels in case-control studies, but the issue is yet unresolved. Mexico has shown an increase in cardiovascular diseases, and some genetic factors may play a role. Our purpose was to evaluate the association between PON1Q192R and L55M polymorphisms and serum lipid profile in a healthy Mexican population. Ninety unrelated male inhabitants from southeastern Mexico with Mayan ascendancy agreed to participate. Demographic characteristics, lifestyle and medical history were obtained by questionnaire. Lipid profile was determined by enzymatic methods, PON1 activity by using paraoxon and phenylacetate and PON1 genotype by real-time PCR. HDL-cholesterol (HDL-C) levels were associated with genotype: 192RR homozygote subjects had lower HDL-C levels than 192QQ homozygotes, and individuals with 192RR and 192QR genotypes had an odds ratio (OR)=7.05 (95% confidence interval (CI)=1.29-38.34) of having HDL-C <60 mg/dL. Individuals with higher paraoxonase activity (>600.18 U/L) had a slight risk (OR=4.9, 95% CI=0.83-22.02) of having HDL-C <60 mg/dL. PON155LM polymorphism was associated with higher LDL-cholesterol. PON1Q192R polymorphism showed a role in modulating lipid profile: 192RR homozygotes showed the least favorable lipoprotein levels.

The induction of cytochrome P450 (CYP) 2E1 in testes and liver and the presence of trifluoroacetylated (TFA) adducts in spermatozoa, testes, liver and plasma were investigated in rats subchronically exposed by inhalation to halothane (15 ppm/4 h/day/5 days/week/9 weeks). After halothane exposure, p-nitrophenol hydroxylase (p-NPH) activity increased 3.2-fold and CYP2E1 apo-protein content 7-fold in testes, whereas in liver, p-NPH increased 2.3-fold and CYP2E1 apoprotein content 1.4-fold. These results suggest a differential inductive effect of halothane on CYP2E1 in these tissues. Moreover, TFA adducts were present in microsomes of testis and liver and in plasma of halothane-treated rats. The immunoblot analysis of testicular microsomes showed two intense TFA protein bands of 63 and 59 kDa, whereas in liver three intense bands of 100, 76 and 63 kDa were observed. Bands of similar molecular weights to those observed in liver were detected in the plasma of halothane-treated animals. In addition, TFA adducts were detected by immunofluorescence in spermatozoa, probably in the acrosome and/or perinuclear theca region, and in the distal tail of spermatozoa. The increase in CYP2E1 apoprotein and p-NPH activity observed in testis and liver microsomes suggests that halothane induces its own biotransformation both hepatically and extrahepatically and in addition, that the nature of the TFA adducts will depend on the proteins present in each tissue. Also, the presence of TFA adducts in spermatozoa may result from the activation of halothane in the reproductive tract. The detailed mechanism of TFA adduct formation and its consequences on the spermatozoa function remain to be fully clarified.

Follicular lymphoma is characterized by the presence of the t(14;18)(q32;q21) chromosomal translocation which juxtaposes the bcl-2 gene at 18q21 with the immunoglobulin heavy chain locus at 14q32. Quantification of t(14;18) carrying cells in FL patients can be achieved by real-time PCR, a highly sensitive technique for evaluating treatment efficacy and minimal residual disease. Despite the many advantages of real-time technology for this purpose, one disadvantage is that current real-time t(14;18) PCR assays amplify a control gene as a normalizer in a separate reaction. Since each PCR reaction has its own kinetics, separate PCR assays for target and control sequences can potentially result in inaccurate quantification of t(14;18)-positive cells. In addition, the real-time t(14;18) PCR assays do not determine the size of the amplified fusion sequence, which is helpful for excluding contamination and is commonly used to demonstrate clonal identity between pre- and post-treatment specimens from a patient. To address these limitations, we designed a multiplex real-time PCR protocol that allows amplification of control and target genes in the same reaction and precise size determination of bcl-2/JH fusion sequences by capillary electrophoresis. This multiplex PCR assay is equally sensitive to previous assays, allows more accurate quantification of bcl-2/JH fusion sequences, and is more convenient.

Adult male rats were exposed to inhale halothane in the following regime: 15 ppm/4 h/5 days/week/9 weeks. Sexual behavior observations and sperm motility test were made before halothane exposure (0 days) and at 15, 30, 45 and 60 days of exposure. Fifteen days after halothane exposure, this anesthetic inhibited the proportion of animals displaying ejaculation. In those animals ejaculating, halothane produced an inhibition of masculine sexual behavior reflected as an increase in the intromission latency, number of mounts and postejaculatory interval. At 30 days after exposure, only an increase in the intromission latency was observed. At 45 and 60 days, the inhibitory effect of halothane on sexual behavior disappeared. Similarly, at 15 and 30 days, but not at 45 or 60 days of halothane exposure, a reduced sperm motility was observed. Such transient effects of halothane suggest the development of tolerance to the inhibitory actions of this anesthetic on sexual behavior and sperm motility. These halothane effects are in line with an inhibition of masculine sexual behavior after stimulation of the GABAergic system.

The ability of chromium (Cr) salts to increase metallothionein (MT) levels in rat liver, kidney and pancreas, and its relationship with the presence of toxic effects are reported here. Rats were injected subcutaneously with 0, 10, 20, 30, 40, or 50 mg K2Cr2O7/kg and sacrificed 24 h later. Total Cr accumulation followed a dose-dependent pattern, levels in kidney being higher than those in liver or pancreas, suggesting different tissue bioavailabilities and accumulation patterns. Cr(IV) administration resulted in a tissue-specific MT induction: pancreas and liver showed five- and 3.5-fold MT increases, respectively; no increase was observed in the kidney. A positive correlation was observed between zinc and MT concentrations in liver, and between total Cr and MT concentrations in pancreas. Serum alpha-amylase activity showed a dose-dependent increase starting from 20 mg/kg, whereas serum glucose levels increased at doses higher than 30 mg/kg. Serum aspartate aminotransferase and alanine aminotransferase activities were increased in a dose-dependent manner, from 20 and 30 mg/kg, respectively. Our results showed that treatment with Cr(VI) can induce MT synthesis in pancreas and suggests a subsequent binding of Cr to MT. Also, pancreas is a target organ for Cr toxicity, and the usefulness of alpha-amylase activity as a sensitive biomarker of Cr toxicity in human exposed populations merits further study.

To identify potential mutations in the gamma-glutamyl carboxylase gene, the sequence of all exons and intron/exon borders was determined in 4 patients from a consanguineous kindred with combined deficiency of all vitamin K-dependent procoagulants and anticoagulants and results were compared with normal genomic sequence. All 4 patients were homozygous for a point mutation in exon 9 that resulted in the conversion of an arginine codon (CTG) to leucine codon (CGG) at residue 394. Screening of this mutation based on introduction of Alu I site in amplified fragment from normal allele but not from the mutated allele showed that 13 asymptomatic members of the kindred were heterozygous for the mutation. The mutation was not found in 340 unrelated normal chromosomes. The segregation pattern of the mutation which is the first reported in the gamma-glutamyl carboxylase gene fits perfectly with phenotype of the disorder and confirms the suggested autosomal recessive pattern of inheritance of combined deficiency of all vitamin K-dependent procoagulants and anticoagulants in this kindred. The mutated carboxylase protein expressed in Drosophila cells was stable but demonstrated threefold reduced activity compared with WT carboxylase, confirming that the L394R mutation results in a defective carboxylase.

Chronic low level lead (Pb) exposure is associated with decrements in renal function in humans, but the molecular mechanisms underlying toxicity are not understood. We investigated cytosolic Pb-binding proteins (PbBP) in kidney of environmentally-exposed humans to identify molecular targets of Pb and elucidate mechanisms of toxicity. This study is unique in that it localized PbBPs based on physiologic Pb that was bound in vivo. Two Pb-binding polypeptides were identified, thymosin beta 4 (T beta 4, 5 kDa) and acyl-CoA binding protein (ACBP, 9 kDa, also known as diazepam binding inhibitor, DBI). These polypeptides, which have not been previously recognized for their metal-binding capabilities, were shown to bind Pb with high affinity (Kd approximately 14 nM) and to account for an estimated > 35% of the total Pb in kidney cortex tissue. Both T beta 4 and ACBP (DBI) occur across animal species from invertebrates to mammals and in all major tissues, serving multiple possible functions (e.g. regulation of actin polymerization, calmodulin-dependent enzyme activity, acyl-CoA metabolism, GABA-A/benzodiazepine receptor modulation, steroidogenesis, etc.). Thus, these data provide the first evidence of specific molecular targets of Pb in kidney of environmentally-exposed humans, and they suggest that low-level Pb toxicity may occur via alteration of T beta 4 and ACBP (DBI) function in renal and other tissues, including the central nervous system.

The human gene for gamma-glutamyl carboxylase is 13 kb in length and contains 15 exons. Transcription starts at a cytosine 217 base pair upstream of the first codon. There are two major transcripts in all tissues examined. They are distinguished by the presence of an Alu sequence in the 3' nontranslated end of the longer species. Relative mRNA levels for 12 bovine tissues are presented.

Parathion (PA) is a phosphorotioate pesticide requiring P-450-mediated oxidations to become activated to paraoxon, or to be metabolised to its less toxic metabolites. On the other hand, sodium arsenite [As(III)] markedly decreases total hepatic P-450 content and dependent monoxygenase activities. Our aim was to determine the effects of As(III) pretreatment on the acute toxicity of PA and its possible relationship with the effects of As(III) on P-450-dependent monooxygenase activities. Adult male Wistar rats were pretreated with As(III) (5.6 mg As(III)/kg, s.c.), and 24 h later given PA (5 to 20 mg/kg, per os). As(III) pretreatment increased the acute toxicity of PA, reducing 38% its median lethal dose (LD50) from 11.68 to 7.21 mg PA/kg. In addition, As(III) pretreatment further decreased the inhibitory effect of PA on brain acetylcholinesterase activity, reducing 33% the median inhibitory dose (ID50) from 3.44 to 2.31 mg PA/kg. whereas As(III) alone had no significant effects. As(III) decreased the P-450 content to 87% of control values, reduced EROD activity to 74% and BROD activity to 41%; PA produced no significant effects on these parameters, whereas the joint administration of As(III)+ PA produced effects similar to those of As(III). PROD activity was reduced to 36% of control value by PA, whereas As(III) alone produced no significant effects. However, As(III) pretreatment apparently protected against the inhibition of CYP2B1-mediated PROD activity produced by PA, since PROD values were similar to those of control animals. Our results also indicated that the increase in PA toxicity caused by As(III) pretreatment, could also be related to the CYP2B2 isoform, since decreases in CYP2B2-dependent BROD activity were observed in both As(III) and As(III) + PA groups, but not in PA-treated animals, suggesting that CYP2B2 is involved in PA detoxification.

This study reports the partial purification and characterization of cytosolic lead binding proteins (PbBPs) in human brain tissue of environmentally Pb-exposed subjects. The isolated proteins were initially characterized based upon the presence of endogenously associated Pb. Following partial purification (Sephadex G-75 and A-25 DEAE anion-exchange chromatography), the isolated PbBPs (contained within a single DEAE peak) showed a single class of high affinity binding sites with an apparent Kd of 10(-9) M, based upon competition assays using radioactive 203Pb and Hill and Scatchard analysis. The presence of endogenously bound Pb with the isolated proteins indicated the association of Pb with the protein(s) in vivo in these environmentally Pb-exposed subjects, since the samples were prepared in an ultraclean lead analysis laboratory. Moreover, the persistence of Pb-protein binding throughout the initial two steps (Sephadex G-75 and A-25 DEAE) of the purification scheme is consistent with the high affinity and stability of binding measured with the radiolead competition assays. The DEAE isolated PbBPs were further purified by denaturing reversed-phase HPLC analysis, resulting in the isolation of two proteins, thymosin beta 4 (5 kDa, pI 5.1) and a second as yet unidentified protein with an approximate molecular mass of 20 kDa and a pI of 5.9. Qualitative 203Pb-binding analysis of these HPLC purified proteins suggested that they may be primarily responsible for the observed Pb binding in the single DEAE peak. Nearly identical results were obtained in brain cytosols from male and female, and young and adult individuals, although further quantitative analyses are needed to investigate possible sex and age relationships. These data are significant because they contribute to a better understanding of the presence of PbBPs in a sensitive target organ for Pb toxicity in humans, suggesting a possible role of these or similar proteins as sensitive biomarkers of Pb exposure and toxicity.

Porphyrin production and excretion and the effects of lead exposure were studied in long-term cultures of adult rat hepatocytes cultured on a feeder layer of 3T3 cells after addition of 5-aminolevulinic acid. Porphyrin excretion into the culture medium showed an irregular profile during the first 10 days, with a maximum increase of 50% at day 4 and at day 10 a value similar to that of day 1. Thereafter, porphyrin excretion decreased progressively to 18% of the initial value after 4 weeks. The cellular porphyrin content, after 7 and 28 days in culture, reached values 3.8 and 2.4-fold higher than the corresponding day 1 value. The exposure to 0.5 and 2.4 microM Pb2+ for up to 28 days produced a biphasic effect on porphyrin excretion. Firstly, there was a progressive decrease up to 81% during the first 6 days of lead exposure and, secondly, this effect was followed by an increase reaching control values at day 15 and of up to 6.7-fold after 22 days of exposure to 2.4 microM Pb2+. Similar changes were observed in cellular porphyrin content. The exposure to 0.5 and 2.4 microM Pb2+ for 2 and 4 weeks also produced morphological alterations and release of cytoplasmic enzymes. Our results show that hepatocytes cultured on 3T3 cells produce and excrete porphyrins for 28 days and that exposure for 4 weeks to micromolar lead concentrations alters these functions and cell morphology and produces cytotoxic effects which are better evaluated by monitoring alterations in porphyrin excretion than by enzyme leakage. They also suggest that this culture system is a useful model for assessing the toxic effects of xenobiotics on the biosynthesis of heme by liver cells.