Chromium Genotoxicity Associated with Respiratory Disease

Chromium existing in the biosphere in prominent two forms Cr (III) and Cr (VI) is a well-studied heavy metal. Cr (III) is considered as non-harmful and necessary element in diet whereas Cr(VI) is extremely toxic exerting various negative health impacts on human and other organisms. Mining activity is must for extracting economic minerals and a large number of people are related to these sites as worker or habitants and a major source of chromium exposure. Present chapter discusses genotoxic nature of chromium considering respiratory disease resulted from chromium exposure. The genotoxicity is illustrated in terms of chromium induced differential expressed genes (DEGs), transcription factors and microRNA regulating the DEGs and their gene ontology.


Introduction
For the growth of economy of a country and improving living status of population, industrial functioning is mandatory which is in other hand associated activities including supply of power, raw materials, processing and discharge of waste.For a major section of industries, power supply is from coal or electricity generated from coal, and the raw materials are various form of ores received from mining.Mine tailing is the fine residual mine dump after completion of mining left with dug out soil, scattered residuals and disturbed ecosystem.The major source of chromium in the mine tailings is the residual ores present in traces not extracted with economic point of view and mineral processing chemicals that are left unattended.Chromium (Cr), a valuable element often finds its utility in metallurgical, chemical, and refractory industries due to its pigment property, hardness and persistence.From environment point of view, chromium exists in three oxidative states, elemental chromium (0) that does not exist naturally, whereas trivalent chromium (Cr III) is rather stable followed by hexavalent chromium (Cr VI) based on the different number of electrons and therefore varied properties [1].Hexavalent chromium is extremely toxic even in low concentration and listed as carcinogenic, hematotoxic and altering genetic material whereas, Cr (III) is regarded as micronutrient in human diet.When Cr is left unattended in mine tailings, it can be transported by natural means to nearby waterbody, added with acid mine drainage, and surrounding ecosystem expanding the circumference of toxicity exposure [2].This chapter emphasises on toxicity of hexavalent chromium in genetic level that influence expression of genes, the transcript factors controlling the differentially expressed genes and finally to find out the major indicating and influenced genetic factors with functional analysis of gene ontology for respiratory units of human.

Source and toxicity of chromium
Toxicity of chromium is directly influenced by the chromium species with valence with number of electrons and thus their properties.The Cr(VI), is a powerful oxidizing agent and plainly toxic to human and other organisms causing adverse effect to blood cells, renal cells, allergic conditions and organs of most part of body failure.Chromium can significantly find its route of exposures through dermal chromium contact in waste sites, inhalation of chromium emissions and ingestion of contaminated water or food grown in chromium contaminated soil.Also, erosion products and emissions from road and cement dust, leather, paints and or any Cr used materials contribute to inhalation of chromium.Dermal ulcers, irritation and sensitization of respiratory/lungs are consecutive result of chromium contact.In the plasma and cells, Cr(VI) readily get reduced to Cr(III), and thereafter excreted in the urine.Trivalent chromium is the form of chromium that is essential to human health and counted as an essential trace mineral in the human diet.Hexavalent chromium is recognised as genotoxic as it can damage genetic information in living cells, causes DNA mutations, and possibly the formation of cancerous tumours.Chromates (chromium salts) formed from hexavalent chromium also finds utilization in manufacture leather products, paints, cement, mortar, anti-corrosives, and other things.They are carcinogenic and allergenic.

Physiologic effects of chromium exposure in respiratory disease
Occupational exposures often include mixed exposure to both Cr(III) and Cr (VI) [3].Chromium compounds, when inhaled, causes respiratory tract irritants, resulting in airway irritation, airway obstruction, and lung, nasal, or sinus cancer.Radiographic analysis from several reports revealed enlargement of the hilar region and lymph nodes [4,5].Consistent associations have been found between employment in the chromium industries and significant risk for respiratory cancer.Moller et al. [6] reported systemic reactions characterised with anaphylactoid reaction in a young welder having chromium (VI) vapor fume exposures.Following an experiment with sodium chromate inhalation at a concentration of 29 μg/m 3 , formation of static urticaria, angioedema and severe bronchospasm simultaneously with plasma histamine rising in threefold was documented and suggested direct positive leukocyte inhibitory factor of sodium chromate.
A number of nasal mucosa injury cases in Cr (VI) exposed workers at concertation of nearly 20 μg/m 3 (against US permissible standard 5 μg/m 3 ) for 5 months to 10 years characterised with inflamed mucosa and ulcerated/perforated septum was recorded in a study with 43 chrome-plating plants and tanneries in Sweden [7,8].Huge number of complaints for nasal irritations was documented in a detail epidemiological study with Tokyo (Japan) housewives residing near chromium slag contaminated construction site [7].U.S has recommended chromate and chromic acid at workplace to be 5 μg/m 3 as permissible standard.Gibb et al. [9] observed that with less than 30 days median time for nasal ulceration diagnosis from first exposure, median Cr (VI) concentration matched the Sweden report.Occupational exposure to Cr(III) has also been associated with respiratory effects.
Persons developed coughing, wheezing, and decreased forced volume after an inhalation exposure to a sample of Cr(III) sulfate [10].Combine effect of Cr(III) and Cr(VI) as total chromium (0.02-0.19 mg total chromium/m3 ) investigated among 60 ferrochromium workers squeezed out subjective symptoms of coughing, wheezing, and dyspnea whereas control remained neutral [11].These symptoms might get puzzled with smoking issue to clarify the accurate problem of the diseases [11].While considering respiratory issue, animals are also often exposed to chromium similar to the human.Henderson et al. [12] in histological examination with exposure of 0.9-25 mg Cr(III) trichloride for 30 min observed alterations in lung tissues associated with mild inflammation.

Chromium-gene interactions in respiratory disease
Comparative toxico-genomics database (CTD, http://ctdbase.org) is a recognised well informed/updated, openly accessible database.It purposes to provide detail knowledge and information about the impacts of exposure of environmental elements (pollutants) on human health.
The core block of the database basically manually curated contains updated information regarding interaction and relationships among chemicals, genes, proteins and their resulted specific disease in terms of functional and pathways to incorporate new hypotheses expressing underlying mechanisms of disease and environmental contamination [13].

Results and discussion
In this work, all Chromium-gene /protein interactions for respiratory disease are downloaded from CTD, in which Chromium-gene /protein interactions associated to the following 04 respiratory disease are selected for further analysis according to MESH ID used in CTD-Lung Neoplasma, Pulmonary Fibrosis and Lung disease.Chromium-gene/protein interactions associated to this respiratory disease are collected for further analysis.According to the reference score on relationships between chemicals-genes, genes-diseases and chemicals-diseases [14], lung neoplasms is recognised as most likely having the maximum connectivity with chromium.(Table 1).From the identified 168 chromium gene with in respiratory disease, 131 genes are unique.

Gene function enrichment analysis
KEGG (http://www.genome.jp/) is a knowledge base for systematic analysis of gene functions, linking genomic information with higher-order functional information [15].For the analysis of Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis, the Database for Annotation, Visualization and Integrated Discovery (DAVID, http://david.abcc.ncifcrf.gov/) is a great option.DAVID provides various functional annotation tools for researchers to understand biological meaning behind large list of genes.[16] Gene ontology (GO) analysis and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway enrichment analysis can be performed for analysing differentially expressed genes (DEGs) at the functional level based on DAVID Bioinformatics Resources 6.8.P < 0.05 as the cut-off criterion.Researchers can upload all DEGs to the online software DAVID to identify overrepresented GO categories and KEGG pathways.The curated genes in CTD for each respiratory disease can be uploaded to DAVID 6.8 Beta (https://david-d.ncif-crf.gov/tools.jsp) with Homo sapiens as the background population [17] for GO analysis.
GO analysis results for Cr toxicity in respiratory organs shows that that chromium interacted genes in respiratory disease are involved in the biological processes (BP) such as positive regulation of gene expression, positive regulation of cell proliferation, response to drug positive regulation of protein phosphorylation.(Table 2) For molecular function (MF), genes are enriched in identical protein binding, enzyme binding, transcription factor binding and protein phosphatase binding (Table 2).In addition, GO cell component (CC) analysis also displayed that the gene are significantly enriched in the extracellular space, protein complex, extracellular region and extracellular exosome (Table 2).
Table 3 contains the most significantly enriched pathways of the chromium interacting genes by KEGG analysis.The interacting genes are enriched in Pathways in cancer, Proteoglycans in cancer, HIF-1 signalling pathway and TNF signalling pathways.

Gene-TFs-miRNAs regulation
The transcription factors (TFs) as well as microRNAs (miRNAs), are recognised for their huge share in transacting and gene regulations with various common logics and regulatory factors for gene regulation in multicellular genomes [18,19].The library of ENCODE and ChEA Consensus TFs from ChIP-X in EnrichR (http://amp.pharm.mssm.edu/Enrichr/[20,21]) can be used for the possible TFs   ZMIZ1 and ESR1 are significant for respiratory disease influencing DEGs.The regulated genes for each of these TFs for chromium toxicity are shown in Table 4 followed by the miRNAs identified for chromium interacting genes involved in the Respiratory diseases in Figure 1.

Comparable chemicals
Information about biological effects of a chemical at genetic level can be extensively extracted from CTD to create new hypotheses with a lot of interaction pathways and networks among genes-contaminants and diseases [22].

Conclusions
Chromium (VI) is a vital toxic environmental pollutant having various sources including mine tailings.This chapter enlighten respiratory disease accelerated as well as caused due to chromium exposure at genetic level following bioinformatics method that leverages curated data from the public database CTD to generate novel sets of information.This strategy does not require a priori knowledge of the toxicant, biological system, or adverse outcome, and it can be used to identify potential molecular and biological intermediary steps that help fill in knowledge gaps connecting chemical exposures with outcomes for environmentally influenced diseases.With the existed data libraries (mainly CTD, GO, pathway, TFs and miRNA relate databases), bioinformatics web-based tools (David and EnrichR), BPs, CCs, MFs, KEEG signal pathways and gene regulation in the chromium-gene-disease networks were presented.In this study, 127 genes are identified as affected by exposure CR(VI), which are majorly regulated by 10 TFs and 10 very high target miRNAs.The Gene-TFs-miRNAs network recognises maximum interacted genes (EFNB2, IGF1R, CYP1B1, INSR, and VEGFA) and TFs (ZMIZ1, NFE2L2, CTCF and RAD21) and miRNAs (hsa-miR-4506, hsa-miR-379, hsa-miR-3529, hsa-miR-4535, hsa-miR-3684, and hsa-miR-409-5p).The significant biological process (positive regulation of gene expression and positive regulation of nitric oxide biosynthetic process), Cellular Component (extracellular space and protein complex) and Molecular Function (identical protein binding and enzyme binding) are influenced by chromium exposures.From pathway analysis of Cr (VI) influence on respiratory disease, maximum of DEGs are identified to be involved in various pathways in cancer (41 nos.) followed by proteoglycans in cancer (30 nos.), and HTLV-I infection (23 nos.) and so on.Comparable contaminants analysis has recognised Mercury, SB 203580, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one and 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one to have maximum common DEGs with Cr (VI) exposure.

Table 1 .
Selected Respiratory diseases and related chromium-interacted gene.
4Trace Elements and Their Effects on Human Health and Diseases and related networks.The TargetScan library in EnrichR can be used for the possible miRNA interaction.TFs are identified to be significantly associated with the genes involved in the respiratory disease.TRIM28, NFE2L2, EGR1 GATA2, PPARG,

Table 2 .
Gene ontology analysis of Cr interacted genes.

Table 3 .
Pathway analysis for the chromium interacting genes related to Respiratory Disease.

Table 4 .
Transcription factors for the chromium interacting genes involved in the Respiratory diseases.Trace Elements and Their Effects on Human Health and Diseases