Occupational exposure to potentially toxic elements alters gene expression profiles in formal and informal Brazilian workers.

Chemical elements, such as toxic metals, have previously demonstrated their ability to alter gene expression in humans and other species. In this study, microarray analysis was used to compare the gene expression profiles of different occupational exposure populations: a) informal workers who perform soldering of jewelry inside their houses (n = 22) in São Paulo (SP) State; and b) formal workers from a steel company (n = 10) in Rio de Janeiro (RJ) state, Brazil. Control participants were recruited from the same neighborhoods without occupational chemical exposure (n = 19 in SP and n = 8 in RJ). A total of 68 blood samples were collected and RNA was extracted and hybridized using an Agilent microarray platform. Data pre-processing, statistical and pathway analysis were performed using GeneSpring software. Different expression was detected by fold-change analysis resulting in 16 up- and 33 down-regulated genes in informal workers compared to the control group. Pathway analysis revealed genes enriched in MAPK, Toll-like receptor, and NF-kappa B signaling pathways, involved in inflammatory and immune responses. In formal workers, 20 up- and 50 down-regulated genes were found related to antimicrobial peptides, defensins, neutrophil degranulation, Fc-gamma receptor-dependent phagocytosis, and pathways associated with atherosclerosis development, which is one of the main factors involved in the progression of cardiovascular diseases. The gene IFI27 was the only one commonly differentially expressed between informal and formal workers and is known to be associated with various types of cancer. In conclusion, differences in gene expression related to occupational exposure are mainly associated with inflammation and immune response. Previous research has identified a link between inflammation and immune responses and the development of chronic diseases, suggesting that prolonged occupational exposures to potentially toxic elements in Brazilian metal workers could lead to negative health outcomes. Further analysis should be carried out to investigate its direct effects and to validate causal associations.

Multi-omics analysis reveals that co-exposure to phthalates and metals disturbs urea cycle and choline metabolism.

This study aimed to evaluate the response of HepaRG cells after co-exposure to phthalates and heavy metals, using a high-dimensional biology paradigm (HDB). Liver is the main metabolism site for the majority of xenobiotics. For this reason, the HepaRG cell line was used as an in vitro model, and cells were exposed to two characteristic mixtures of phthalates and heavy metals containing phthalates (DEHP, DiNP, BBzP) and metals (lead, methylmercury, total mercury) in a concentration-dependent manner. The applied chemical mixtures were selected as the most abundant pollutants in the REPRO_PL and PHIME cohorts, which were studied using the exposome-wide approach in the frame of the EU project HEALS. These studies investigated the environmental causation of neurodevelopmental disorders in neonates and across Europe. The INTEGRA computational platform was used for the calculation of the effective concentrations of the chemicals in the liver through extrapolation from human biomonitoring data and this dose (and a ten-times higher one) was applied to the hepatocyte model. Multi-omics analysis was performed to reveal the genes, proteins, and metabolites affected by the exposure to these chemical mixtures. By extension, we could detect the perturbed metabolic pathways. The generated data were analyzed using advanced bioinformatic tools following the HEALS connectivity paradigm for multi-omics pathway analysis. Co-mapped transcriptomics and proteomics data showed that co-exposure to phthalates and heavy metals leads to perturbations of the urea cycle due to differential expression levels of arginase-1 and -2, argininosuccinate synthase, carbamoyl-phosphate synthase, ornithine carbamoyltransferase, and argininosuccinate lyase. Joint pathway analysis of proteomics and metabolomics data revealed that the detected proteins and metabolites, choline phosphate cytidylyltransferase A, phospholipase D3, group XIIA secretory phospholipase A2, α-phosphatidylcholine, and the a 1,2-diacyl-sn-glycero-3-phosphocholine, are responsible for the homeostasis of the metabolic pathways phosphatidylcholine biosynthesis I, and phospholipases metabolism. The urea, phosphatidylcholine biosynthesis I and phospholipase metabolic pathways are of particular interest since they have been identified also in human samples from the REPRO_PL and PHIME cohorts using untargeted metabolomics analysis and have been associated with impaired psychomotor development in children at the age of two. In conclusion, this study provides the mechanistic evidence that co-exposure to phthalates and metals disturb biochemical processes related to mitochondrial respiration during critical developmental stages, which are clinically linked to neurodevelopmental perturbations.

Unraveling the blood transcriptome after real-life exposure of Wistar-rats to PM2.5, PM1 and water-soluble metals in the ambient air.

Exposure to particulate matter (PM) is one of the most important environmental issues in Europe with major health impact. Various sizes of PM are suspended in the atmosphere and contributes to ambient air pollution. The current study aimed to explore the differential gene expression in blood, and the effect on the respective biological signaling pathways in Wistar rats, after exposure to PM2.5 and PM1 ambient air particles for an eight-week period. A control group was included with animals breathing non-filtered atmospheric air. In parallel, filtered PM2.5 and PM1 was collected in separate samplers. The results after whole genome microarray analysis showed 23 differentially expressed genes (DEGs) between control and PM2.5 group. In addition, pairwise comparison between control and PM1 group displayed 5635 DEGs linked to 69 biological pathways involved in inflammatory response, cell cycle and carcinogenicity. The smaller the size of the inhaled particles, the more gene alterations are triggered compared to non-filtered air group. More specifically, in inflammation signaling procedures differentially regulated gene expression was shown for interleukin-4 (IL-4), IL-7, IL-1, IL-5, IL-9, IL-6 and IL-2. We have identified that RASGFR1, TRIM65, TRIM33, PLEKHB1, CAR4, S100A8, S100A9, ALPL, NP4 and the PROK2 genes are potential targets for the development of adverse outcome pathways (AOPs) due to "real-life" exposure of Wistar rats. Particle measurements during the exposure period showed elevated concentrations of Fe, Mn and Zn in both PM1 and PM2.5 filter fractions, and of Cu in PM2.5. In addition, water-soluble concentration of metals showed significant differences between PM1 and PM2.5 fractions for V, Zn, As, Pb and Mn. In summary, in this study specific gene biomarkers of exposure to ambient air have been identified and heavy metals that are possibly linked to their altered regulation have been found. The results of this research will pave the way for the development of novel AOPs concerning the health effects of the environmental pollution.

Immunity raised by recent European subtype 1 PRRSV strains allows better replication of East European subtype 3 PRRSV strain Lena than that raised by an older strain.

Stable spatial distribution of porcine reproductive and respiratory syndrome (PRRSV)-1 subtypes in Europe is accompanied by a strong population immunity induced by local PRRSV strains. In the present study, it was examined if the immunity induced by three West European subtype 1 PRRSV strains (2007 isolate 07V063 and 2013 isolates 13V091 and 13V117) offers protection against the highly virulent East European subtype 3 PRRSV strain Lena. The number of fever days was greater (p < 0.05) in the control group (7.6 ± 1.7 days) compared to the immune groups (07V063-immune: 4.0 ± 1.2 days, 13V091-immune: 4.6 ± 1.1 days, 13V117-immune: 4.0 ± 2.9 days). In all groups, protection was characterized by reduction (p < 0.05) of AUC values of nasal shedding (control: 14.6, 07V063-immune: 3.4, 13V091-immune: 8.9, 13V117-immune: 8.0) and viremia (control: 28.1, 07V063-immune: 5.4, 13V091-immune: 9.0, 13V117-immune: 8.3). Reduction of respiratory disease, nasal shedding (mean AUC and mean peak values) and viremia (mean AUC and mean peak values) was more pronounced in 07V063-immune (p < 0.05) than in 13V091-immune and 13V117-immune animals. Inoculation with subtype 1 PRRSV strains caused priming of the Lena-specific virus neutralization antibody response. Upon challenge with Lena, we observed a very strong serological booster effect for neutralizing antibodies against strains used for the first inoculation. Our results indicate that inoculation with subtype 1 PRRSV strains can partially protect against antigenically divergent subtype 3 strains. The lower protection level elicited by recently isolated subtype 1 PRRSV strains may impair the outcome of the spatial expansion of subtype 3 strains from East Europe to West Europe.

Replication characteristics of eight virulent and two attenuated genotype 1 and 2 porcine reproductive and respiratory syndrome virus (PRRSV) strains in nasal mucosa explants.

Porcine reproductive and respiratory syndrome virus (PRRSV) can spread in between pigs via contact and airborne route. It was shown before that the highly pathogenic PRRSV strain Lena was able to replicate 10-100 times more in the nasal mucosa compared to the low pathogenic PRRSV strain LV. In this work, the replication characteristics of four type 1 (LV, 07V063, 08VA, 13V091), three type 2 (VR2332, MN-184, VN) and two attenuated (MLV-DV, MLV-VR2332) PRRSV strains were studied. After 72hpi, mean virus titers reached 10(4.5-4.8) TCID50/ml for LV and 08VA, 10(5.2-5.4) TCID50/ml for VR2332 and Lena, and 10(5.8-6.3) TCID50/ml for 07V063, 13V091, MN-184 and VN strains, whereas attenuated strains remained below detection limit. The mean number of PRRSV-positive cells/mm(2) at 72hpi was 1.1 and 1.3 for the attenuated strains and LV, 13.3 for 08VA, 23.5 and 29.3 for VR2332 and 07063, 31.1 and 33.8 for 13V091 and Lena, and, 39.1 and 59.2 for MN-184 and VN respectively. All the LV and MLV-LV infected cells were Sn(+), whereas all other strains also infected Sn(-) macrophages. In conclusion, (i) based on the virus shedding in the respiratory explants, PRRSV strains can be categorized as poor (MLV-DV, MLV-VR2332, LV, 08VA), moderate (Lena, VR2332) and strong (07V063, 13V091, MN-184, VN) secretors, and (ii) based on the number of infected cells isolates can be categorized as low (MLV-DV, MLV-VR2332, LV), moderately (08VA, VR2332), highly (07V063, Lena, 13V091) and hyper (MN-184, VN) virulent in the nasal mucosa.

Different clinical, virological, serological and tissue tropism outcomes of two new and one old Belgian type 1 subtype 1 porcine reproductive and respiratory virus (PRRSV) isolates.

In this study, the pathogenic behavior of PRRSV 13V091 and 13V117, isolated in 2013 from two different Belgian farms with enzootic respiratory problems shortly after weaning in the nursery, were compared with the Belgian strain 07V063 isolated in 2007. Full-length genome sequencing was performed to identify their origin. Twelve weeks-old pigs were inoculated intranasally (IN) with 13V091, 13V117 or 07V063 (9 pigs/group). At 10 days post inoculation (dpi), 4 animals from each group were euthanized and tissues were collected for pathology, virological and serological analysis. 13V091 infection resulted in the highest respiratory disease scores and longest period of fever. Gross lung lesions were more pronounced for 13V091 (13%), than for 13V117 (7%) and 07V063 (11%). The nasal shedding and viremia was also most extensive with 13V091. The 13V091 group showed the highest virus replication in conchae, tonsils and retropharyngeal lymph nodes. 13V117 infection resulted in the lowest virus replication in lymphoid tissues. 13V091 showed higher numbers of sialoadhesin(-) infected cells/mm(2) in conchae, tonsils and spleen than 13V117 and 07V063. Neutralizing antibody response with 07V063 was stronger than with 13V091 and 13V117. It can be concluded that (i) 13V091 is a highly pathogenic type 1 subtype 1 PRRSV strain that replicates better than 07V063 and 13V117 and has a strong tropism for sialoadhesin(-) cells and (ii) despite the close genetic relationship between 13V117 and 07V063, 13V117 has an increased nasal replication and shedding, but a decreased replication in lymphoid tissues compared to 07V063.

Replication characteristics of porcine reproductive and respiratory syndrome virus (PRRSV) European subtype 1 (Lelystad) and subtype 3 (Lena) strains in nasal mucosa and cells of the monocytic lineage: indications for the use of new receptors of PRRSV (Lena).

Recently, it has been demonstrated that subtype 3 strains of European type porcine reproductive and respiratory syndrome virus (PRRSV) are more virulent/pathogenic than subtype 1 strains. This points to differences in the pathogenesis. In the present study, a new polarized nasal mucosa explant system was used to study the invasion of the low virulent subtype 1 PRRSV strain Lelystad (LV) and the highly virulent subtype 3 PRRSV strain Lena at the portal of entry. Different cell types of the monocytic lineage (alveolar macrophages (PAM), cultured blood monocytes and monocyte-derived dendritic cells (moDC)) were enclosed to examine replication kinetics of both strains in their putative target cells. At 0, 12, 24, 48 and 72 hours post inoculation (hpi), virus production was analyzed and the infected cells were quantified and identified. Lena replicated much more efficiently than LV in the nasal mucosa explants and to a lesser extent in PAM. Differences in replication were not found in monocytes and moDC. Confocal microscopy demonstrated that for LV, almost all viral antigen positive cells were CD163⁺Sialoadhesin (Sn)⁺, which were mainly located in the lamina propria of the respiratory mucosa. In Lena-infected nasal mucosa, CD163⁺Sn⁺, CD163⁺Sn⁻ and to a lesser extent CD163⁻Sn⁻ monocytic subtypes were involved in infection. CD163⁺Sn⁻ cells were mostly located within or in the proximity of the epithelium. Our results show that, whereas LV replicates in a restricted subpopulation of CD163⁺Sn⁺ monocytic cells in the upper respiratory tract, Lena hijacks a broader range of subpopulations to spread within the mucosa. Replication in CD163⁺Sn⁻ cells suggests that an alternative entry receptor may contribute to the wider tropism of Lena.