Elevated Levels of Cytotoxicity, Cytokines, and Anti-SARS-CoV-2 Antibodies in Mild Cases of COVID-19.

Current evidence shows higher production of cytokines and antibodies against severe acute respiratory coronavirus 2 (SARS-CoV-2) in severe and critical cases of Coronavirus Disease 2019 (COVID-19) in comparison with patients with moderate or mild disease. A recent hypothesis proposes an important role of genotoxicity and cytotoxicity in the induction of the cytokine storm observed in some patients at later stages of the disease. Interestingly, in this study, we report significantly higher levels of interleukin (IL)-1ß, IL-6, MCP-1, and IL-4 cytokines in mild COVID-19 patients versus severe cases, as well as a high frequency of karyorrhexis (median [Me] = 364 vs. 20 cells) and karyolysis (Me = 266 vs. 52 cells) in the mucosal epithelial cells of both groups of patients compared with uninfected individuals. Although we observed higher levels of anti-SARS-CoV-2 IgM and IgG antibodies in COVID-19 patients, IgM antibodies were significantly higher only in mild cases, for the N and the S viral antigens. High levels of IgG antibodies were observed in both mild and severe cases. Our results showed elevated concentrations of proinflammatory and anti-inflammatory cytokines in mild cases, which may reflect an active innate immune response and could be related to the higher IgM and IgG antibody levels found in those patients. In addition, we found that SARS-CoV-2 infection induces cytotoxic damage in the oral mucosa, highlighting the importance of studying the genotoxic and cytotoxic events induced by infection and its role in the pathophysiology of COVID-19.

Transcriptome-Wide Analysis of Low-Concentration Exposure to Bisphenol A, S, and F in Prostate Cancer Cells.

Bisphenol A (BPA) is a ubiquitous synthetic compound used as a monomer in the production of polycarbonate plastics and epoxy resins. Even at low doses, BPA has been associated with the molecular progression of diseases such as obesity, metabolic syndrome, and hormone-regulated cancers due to its activity as an endocrine-disrupting chemical (EDC). Consequently, the use of BPA has been regulated worldwide by different health agencies. BPA structural analogs such as bisphenol S and bisphenol F (BPS and BPF) have emerged as industrial alternatives, but their biological activity in the molecular progression of cancer remains unclear. Prostate cancer (PCa) is a hormone-dependent cancer, and the role of BPA structural analogs in PCa progression is still undescribed. In this work, we use an in vitro model to characterize the transcriptomic effect of low-concentration exposure to bisphenol A, S, or F in the two main stages of the disease: androgen dependency (LNCaP) and resistance (PC-3). Our findings demonstrated that the low concentration exposure to each bisphenol induced a differential effect over PCa cell lines, which marks the relevance of studying the effect of EDC compounds through all the stages of the disease.

Prenatal Particulate Matter (PM) Exposure and Natriuretic Peptides in Newborns from Mexico City.

(1) Background: The aim of this study was to assess associations between particulate matter (PM) exposure and natriuretic peptide concentrations in cord blood from newborns. (2) Methods: we conducted a cross-sectional study in Mexico City with 101 pregnant women from CIMIGEN Hospital. Atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) were measured in plasma from cord blood in 51 newborns by ELISA. We estimated PM exposure (PM2.5 and PM10) at first, second and third trimester of pregnancy. (3) Results: The median and interquartile range for ANP, BNP and CNP plasma concentrations were 66.71 (46.92-80.23), 98.23 (73.64-112.30) and 1129.11 (944.10-1452.02) pg/mL, respectively. PM2.5 and PM10 levels for the whole pregnancy period were 22.2 µg/m3 and 41.63 µg/m3, respectively. Employing multivariable linear regression models adjusted for maternal age, newborn sex, smoking before pregnancy, maternal occupation and newborns' length and height, we observed a 2.47 pg/mL (95%CI: -4.67, -0.27) decrease in BNP associated with PM2.5 exposure during second trimester. Adjusted for the same set of confounders, third trimester PM10 exposure was inversely associated with ANP concentrations (beta estimate: -0.90; 95% CI: -1.80, -0.03). Neither PM10 nor PM2.5 were associated with CNP at any trimester of pregnancy. (4) Conclusions: Prenatal exposure to particulate matter was associated with ANP and BNP decrease in newborns.

Maternal overnutrition before and during pregnancy induces DNA damage in male offspring: A rabbit model.

Using a rabbit model, we investigated whether maternal intake of a high-fat and high-carbohydrate diet (HFCD) before and during pregnancy induces an increase in micronuclei frequency and oxidative stress in offspring during adulthood. Female rabbits received a standard diet (SD) or HFCD for two months before mating and during gestation. The offspring from both groups were nursed by foster mothers fed SD until postnatal day 35. After weaning, all the animals received SD until postnatal day 440. At postnatal day 370, the frequency of micronuclei in peripheral blood reticulocytes (MN-RETs) increased in the male offspring from HFCD-fed mothers compared with the male offspring from SD-fed mothers. Additionally, fasting serum glucose increased in the offspring from HFCD-fed mothers compared with the offspring from SD-fed mothers. At postnatal day 440, the offspring rabbits were challenged with HFCD or continued with SD for 30 days. There was an increase in MN-RET frequency in the male rabbits from HFCD-fed mothers, independent of the type of challenging diet consumed during adulthood. The challenge induced changes in serum cholesterol, LDL and HDL that were influenced by the maternal diet and offspring sex. We measured malondialdehyde in the liver of rabbits as an oxidative stress marker after diet challenge. Oxidative stress in the liver only increased in the female offspring from HFCD-fed mothers who were also challenged with this same diet. The data indicate that maternal overnutrition before and during pregnancy is able to promote different effects depending on the sex of the animals, with chromosomal instability in male offspring and oxidative stress and hypercholesterolemia in female offspring. Our data might be important in the understanding of chronic diseases that develop in adulthood due to in utero exposure to maternal diet.

Ability of low contents of biosorbents to bind the food carcinogen aflatoxin B1in vitro.

In vitro experiments were conducted to evaluate the effectiveness of two new biosorbents (lettuce and field horsetail) in removing aflatoxin B1 (AFB1). Formosa firethorn was used as reference material. The adsorption of AFB1 (190 ng/mL) was investigated at two sorbent contents (0.5% and 0.1% w/v) and three pHs (2, 5, and 7). Batch experiments were performed at 40 °C for 2 h. Several methodologies were used to characterize the nature of the biosorbent-AFB1 interaction. In general, when using biosorbents at 0.5% w/v, AFB1 was well adsorbed by the three tested biomaterials (70 to 100%). Furthermore, with the lowest biosorbent content (0.1% w/v), significant AFB1 adsorption efficiencies were attained at pH 5 (33 to 50%). Nevertheless, at pH 7, lettuce showed the highest ability against AFB1 removal (95%). Further characterization of the AFB1-loaded biosorbents demonstrated that chemical and physical mechanisms were involved in the adsorption process.

Particulate matter-associated micronuclei frequencies in maternal and cord blood lymphocytes.

Studies associate particulate matter (PM) exposure with pulmonary, cardiovascular, and neurologic diseases. Elevated levels of coarse (PM10) and fine (PM2.5) PM have been reported in the Mexico City metropolitan area during the last two decades. There is limited information if these conditions affect newborns. We associated maternal exposure to PM reported by the monitoring stations considering the place of residence of each participant with the presence of genotoxic damage (cytome analysis) in maternal and umbilical cord blood (UCB) lymphocytes. Eighty-four healthy women in their last quarter of pregnancy met the inclusion criteria. Each volunteer exposure was estimated according to the average PM2.5 and PM10 levels during the last month of gestation. The micronuclei (MN) frequencies in UCB lymphocyte cultures ranged between 0 and 9. They also showed lower cell proliferation indexes than their mothers. There was a strong correlation between the maternal and the UCB MN frequency (ρ = 0.3767, P = 0.0002). Multiple regression analysis including PM10 and PM2.5 levels, maternal age, and occupation, showed a significant and positive association between UCB MN frequency and PM2.5. A statistically significant increase in the MN frequency in both maternal and UCB lymphocytes was observed in samples obtained during the dry season (higher PM levels) as compared with the MN frequency in blood samples obtained during the rainy season (lower PM levels). These results suggest that PM, mainly PM2.5 , can cross the placenta causing DNA damage in fetal cells which may increase the potential for diseases during childhood or adult life. Environ. Mol. Mutagen. 60:421-427, 2019. © 2019 Wiley Periodicals, Inc.

Micronucleus frequency is correlated with antioxidant enzyme levels in workers occupationally exposed to pesticides.

Oxidative stress can cause DNA damage leading to nuclear anomalies such as micronuclei (MN). Antioxidant enzymes involved in protection against intracellular oxidative stress include glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutase (SOD), and catalase (CAT). Pesticide exposure induces oxidative stress and alters antioxidant defense mechanisms, including detoxification and scavenger enzymes. The aim of this study was to evaluate MN frequency in workers occupationally exposed to pesticides and their relationship with antioxidant enzyme activities. A cross-sectional study was conducted in 201 individuals, some of whom were dedicated to the spraying of pesticides. The cytokinesis-block micronucleus (CBMN) assay was conducted, and the activities of GPx, GR, SOD, and CAT were determined. The geometric mean (GM) of MN was 5.4 (1-26 MN). The GM for the antioxidant enzymes was 198.68 U/mL for GPx, 38.96 U/g Hb for GR, 94.78 U/mL for SOD, and 69.77 U/g Hb for CAT. There was a lower MN frequency in males than that in females, and a higher nuclear index. In addition, age affected MN frequency. There was a negative correlation between MN frequency and GPx activity, but a positive one between MN frequency and GR activity. These findings suggest the involvement of GPx in MN frequency.

Phytochemical study and evaluation of cytotoxic and genotoxic properties of extracts from Clusia latipes leaves

Abstract Some species of the Clusia genus have been shown to have important biomedical properties, including the ability to inhibit tumor growth in vitro and the usefulness for skin care. In this study, we examined the cytotoxic effect of hexane, ethyl acetate and methanol extracts from Clusia latipes Planch. & Triana, Clusiaceae, leaves on survival of human prostate cancer cells (PC-3), colon cancer cells (RKO), astrocytoma cells (D-384), and breast cancer cells (MCF-7). The ethyl acetate extract displayed the most substantial cytotoxic effect. However, using a Comet assay, we observed that the hexane extract induced a genotoxic effect (DNA damage) on human lymphocytes in an in vitro model. Chromatographic purification of the C. latipes hexane extract led to the isolation and identification of friedelin, friedolan-3-ol, and hesperidin as active cytotoxic compounds in hexane extract, while β-amyrine was identified as an active cytotoxic compound in the ethyl acetate extract of C. latipes, thereby supporting further studies of the molecular mechanisms underlying the effect of these secondary metabolites on cancer cell survival.

Arsenic exposure and calpain-10 polymorphisms impair the function of pancreatic beta-cells in humans: a pilot study of risk factors for T2DM.

The incidence of type 2 diabetes mellitus (T2DM) is increasing worldwide and diverse environmental and genetic risk factors are well recognized. Single nucleotide polymorphisms (SNPs) in the calpain-10 gene (CAPN-10), which encodes a protein involved in the secretion and action of insulin, and chronic exposure to inorganic arsenic (iAs) through drinking water have been independently associated with an increase in the risk for T2DM. In the present work we evaluated if CAPN-10 SNPs and iAs exposure jointly contribute to the outcome of T2DM. Insulin secretion (beta-cell function) and insulin sensitivity were evaluated indirectly through validated indexes (HOMA2) in subjects with and without T2DM who have been exposed to a gradient of iAs in their drinking water in northern Mexico. The results were analyzed taking into account the presence of the risk factor SNPs SNP-43 and -44 in CAPN-10. Subjects with T2DM had significantly lower beta-cell function and insulin sensitivity. An inverse association was found between beta-cell function and iAs exposure, the association being more pronounced in subjects with T2DM. Subjects without T2DM who were carriers of the at-risk genotype SNP-43 or -44, also had significantly lower beta-cell function. The association of SNP-43 with beta-cell function was dependent on iAs exposure, age, gender and BMI, whereas the association with SNP-44 was independent of all of these factors. Chronic exposure to iAs seems to be a risk factor for T2DM in humans through the reduction of beta-cell function, with an enhanced effect seen in the presence of the at-risk genotype of SNP-43 in CAPN-10. Carriers of CAPN-10 SNP-44 have also shown reduced beta-cell function.

The reduction of Calpain-10 expression is associated with risk polymorphisms in obese children.

Excessive weight gain and obesity are major public health concerns. Childhood obesity is growing at an alarming rate. Polymorphisms in the Calpain-10 gene and the reduced expression of this gene in muscle cells and adipocytes have been associated with an increased risk of type 2 diabetes mellitus in several populations. In the present study, we explored the contribution of Calpain-10 in the development of metabolic impairment in childhood. We evaluated the presence of risk polymorphisms in the CAPN10 gene (SNP-44, SNP-43, InDel-19 and SNP-63) and the associated changes in the Calpain-10 mRNA levels in a pediatric population. A total of 161 Mexican children between 4 and 18 years old were included in this study. This population was classified into three groups according to international growth references: healthy weight (HW), overweight (OW) and obese (OB). Association studies of the anthropometric data, clinical values, genotyping and expression assays showed a decrease in the Calpain-10 mRNA and protein expression in the OW and OB groups with respect to the HW group. This decrease in the Calpain-10 mRNA expression was more evident in individuals homozygous for SNP-44 (T/T) and InDel-19 (3/3), alone (p<0.001 and p=0.015, respectively) or in combination (p=0.017). These polymorphisms were also associated with elevated BMI, weight percentiles, z-scores, waist circumferences, fasting glucose levels and beta cell functions in the OW and OB groups (p<0.05). Moreover, our results indicate a statistically significant decrease in the expression of the 75-kDa Calpain-10 isoform in the OW+OB group. The presence of polymorphisms and alterations in the expression of the CAPN10 gene at early ages might result in metabolic impairment in adulthood and should be further investigated.

Suppression of p53 and p21CIP1/WAF1 reduces arsenite-induced aneuploidy.

Aneuploidy and extensive chromosomal rearrangements are common in human tumors. The role of DNA damage response proteins p53 and p21(CIP1/WAF1) in aneugenesis and clastogenesis was investigated in telomerase immortalized diploid human fibroblasts using siRNA suppression of p53 and p21(CIP1/WAF1). Cells were exposed to the environmental carcinogen sodium arsenite (15 and 20 microM), and the induction of micronuclei (MN) was evaluated in binucleated cells using the cytokinesis-block assay. To determine whether MN resulted from missegregation of chromosomes or from chromosomal fragments, we used a fluorescent in situ hybridization with a centromeric DNA probe. Micronuclei were predominantly of clastogenic origin in control cells regardless of p53 or p21(CIP1/WAF1) expression. MN with centromere signals in cells transfected with NSC siRNA or Mock increased 30% after arsenite exposure, indicating that arsenite induced aneuploidy in the tGM24 cells. Although suppression of p53 increased the fraction of arsenite-treated cells with MN, it caused a decrease in the fraction with centromeric DNA. Suppression of p21(CIP1/WAF1) like p53 suppression decreased the fraction of MN with centromeric DNA. Our results suggest that cells lacking normal p53 function cannot become aneuploid because they die by mitotic arrest-associated apoptosis, whereas cells with normal p53 function that are able to exit from mitotic arrest can become aneuploid. Furthermore, our current results support this role for p21(CIP1/WAF1) since suppression of p21(CIP1/WAF1) caused a decrease in aneuploidy induced by arsenite, suggesting that p21(CIP1/WAF1) plays a role in mitotic exit.

Relationship between micronuclei formation and p53 induction.

Human exposure to multiple chemicals compromises the integrity of genetic material. Hence, it is essential to determine the extent of DNA damage induced by xenobiotics. In cell lines, the induction of p53 expression in response to treatments with DNA-damaging agents has been proposed as a tool for the detection of genotoxic damage, although a direct correlation between a marker of chromosomal damage and p53 expression has not previously been studied. The micronucleus assay is a widely used genotoxicity test that has been shown to detect structural and numerical chromosomal damage. The present study was designed to characterize the relationship between micronuclei and p53 induction. RKO cells were cultured and treated with non-cytotoxic concentrations of colchicine, vinblastine, bleomycin or arsenic. Mannitol and clofibrate, which are non-genotoxic chemicals, were also included. The frequency of micronuclei was evaluated using the cytokinesis-block assay, and p53 induction was measured by Western blot assay. Our data showed that a significant induction of micronuclei and of p53 protein occurred only with the genotoxic chemicals. No differences in p53 induction were associated with the clastogenic or aneuplodogenic potential of the chemical exposure. The linear regression analysis revealed a direct relationship between p53 levels and the induction of micronuclei (p=0.0001, r(2)=0.9372), indicating that the level of p53 is associated with chromosomal damage.

Arsenite reduces insulin secretion in rat pancreatic beta-cells by decreasing the calcium-dependent calpain-10 proteolysis of SNAP-25.

An increase in the prevalence of type 2 diabetes has been consistently observed among residents of high arsenic exposure areas. We have previously shown that in rat pancreatic beta-cells, low arsenite doses impair the secretion of insulin without altering its synthesis. To further study the mechanism by which arsenite reduces insulin secretion, we evaluated the effects of arsenite on the calcium-calpain pathway that triggers insulin exocytosis in RINm5F cells. Cell cycle and proliferation analysis were also performed to complement the characterization. Free [Ca2+]i oscillations needed for glucose-stimulated insulin secretion were abated in the presence of subchronic low arsenite doses (0.5-2 microM). The global activity of calpains increased with 2 microM arsenite. However, during the secretion of insulin stimulated with glucose (15.6 mM), 1 microM arsenite decreased the activity of calpain-10, measured as SNAP-25 proteolysis. Both proteins are needed to fuse insulin granules with the membrane to produce insulin exocytosis. Arsenite also induced a slowdown in the beta cell line proliferation in a dose-dependent manner, reflected by a reduction of dividing cells and in their arrest in G2/M. Data obtained showed that one of the mechanisms by which arsenite impairs insulin secretion is by decreasing the oscillations of free [Ca(2+)]i, thus reducing calcium-dependent calpain-10 partial proteolysis of SNAP-25. The effects in cell division and proliferation observed with arsenite exposure can be an indirect consequence of the decrease in insulin secretion.

Micronuclei induced by airborne particulate matter from Mexico City.

Particulate air pollution is an important environmental health risk. In the present study, we have investigated the ability of chemically characterized water and organic-soluble extracts of PM(10) from two different regions of Mexico City to induce micronuclei in a human epithelial cell line. We also evaluated the association between the chemical characteristics of the PM and its genotoxicity. The airborne particulate samples were collected from an industrial and a residential region; a Hi-Vol air sampler was used to collect PM(10) on glass fiber filters. PM mass was determined by gravimetric analysis of the filters. One section of each PM(10) filter was agitated either with deionized water to extract water-soluble compounds or with dichloromethane to prepare organic-soluble compounds. The chemical composition of the extracts was determined by ion and gas chromatography and atomic adsorption spectroscopy. A549-human alveolar epithelial cells were exposed to different concentrations of PM(10) extracts and the cytokinesis blocked micronucleus assay was performed to measure DNA damage. Even though the industrial region had a higher PM concentration, higher amounts of metals and PAHs were found in the residential area. Both industrial and residential extracts induced a significant concentration-related increase in the micronuclei frequency. The PM(10) water-soluble industrial extract induced significantly more micronuclei than the one of the residential region; inversely, the organic residential extract induced more micronuclei than the one from the industrial region. The association between the induction of micronuclei and the chemical components obtained by the comparative analysis of standardized regression coefficients showed that cadmium and PAHs were significantly associated with micronuclei induction. Data indicate that water-soluble metals and the organic-soluble fraction of PM(10) are both important in the production of micronuclei. Effects observed, point to the risk of PM exposure and shows the need of integrative studies.

p53 codon 72 polymorphism, DNA damage and repair, and risk of non-melanoma skin cancer.

A very common polymorphism of p53, that of codon 72, codes either for a proline (P72) or an arginine (R72). The two alleles differ in their biological properties: P72 is a stronger inducer of p21, while R72 induces 5-10 times more apoptosis. It is not known, however, whether this polymorphism influences genome stability. The influence of p53 codon 72 polymorphism on cancer risk has been studied for different types of cancer with mixed and inconsistent results. With respect to sporadic non-melanoma skin cancer (NMSC), there are few studies, with small sample sizes, and none in a Latinoamerican population. These studies have found no association between p53 genotype at codon 72 and NMSC. We analyzed whether p53 codon 72 genotype influences genomic stability and the sensitivity of cells to UVB. We also carried out a case-control study of NMSC in a Mexican population which included 204 BCC cases, 42 SCC cases, and 238 controls. There was no association between p53 genotype and basal levels of DNA damage, oxidative DNA damage sensitivity, or DNA repair capacity. R72 dominantly increased the in vitro sensitivity of cells to UVB-induced apoptosis. There was no significant association either between p53 genotype and basal cell carcinoma (BCC), squamous cell carcinoma (SCC) or both combined.

p53 expression in circulating lymphocytes of non-melanoma skin cancer patients from an arsenic contaminated region in Mexico. A pilot study.

Arsenic is a common environmental toxicant and epidemiological studies associate arsenic exposure with various pathologic disorders and several types of cancer. Skin cancers are the most common arsenic-induced neoplasias and the prevalence of skin lesions has been reported to be significantly elevated in individuals exposed to arsenic via drinking water in Mexico. Being lymphocytes the main cells used for human monitoring, we evaluated the expression of p53 protein in the lymphocytes from 44 healthy individuals and 19 samples from individuals living in a chronic arsenicism endemic region. Of the latter group, 12 individuals had non-melanoma skin cancer and 9 of them expressed p53 in the circulating lymphocytes, whereas only one of the 7 non-cancer arsenic exposed individuals expressed it. In the healthy non-arsenic exposed group only one from 44 individuals expressed the protein. These results suggest a clear relationship between non-melanoma skin cancer and p53 expression in circulating lymphocytes. p53 expression in circulating lymphocytes should be evaluated as a potential biomarker of effect or susceptibility.