Isocyanates, a group of low molecular weight aromatic and aliphatic compounds containing the isocyanate group (-NCO), are important raw materials with diverse industrial applications; however, pathophysiological implications resulting from occupational and accidental exposures of these compounds are hitherto unknown. Although preliminary evidence available in the literature suggests that isocyanates and their derivatives may have deleterious health effects including immunotoxicity, but molecular mechanisms underlying such an effect have never been addressed. The present study was carried out to assess the immunotoxic response of methyl isocyanate (MIC) on cultured human lymphocytes isolated from healthy human volunteers. Studies were conducted to evaluate both dose-dependent and time-course response (n = 3), using N-succinimidyl N-methylcarbamate, a surrogate chemical substitute to MIC. Evaluation of DNA damage by ataxia telangiectasia mutated (ATM) and gamma H2AX protein phosphorylation states; measure of apoptotic index through annexin-V/PI assay, apoptotic DNA ladder assay, and mitochondrial depolarization; induction of oxidative stress by CM-H2DCFDA and formation of 8-hydroxy-2' deoxy guanosine; levels of antioxidant defense system enzyme glutathione reductase; and multiplex cytometric bead array analysis to quantify the secreted levels of inflammatory cytokines, interleukin-8, interleukin-1beta, interleukin-6, interleukin-10, tumor necrosis factor, and interleukin-12p70 parameters were carried out. The results of the study showed a dose- and time-dependent response, providing evidence to hitherto unknown molecular mechanisms of immunotoxic consequences of isocyanate exposure at a genomic level. We anticipate these data along with other studies reported in the literature would help to design better approaches in risk assessment of occupational and accidental exposure to isocyanates.

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Isocyanates (R--N==C==O), one of the highly reactive industrial intermediates, possess the capability to modulate the bio-molecules by forming toxic metabolites and adducts which may cause adverse health effects. Some of their toxic degradations have previously been unknown and overlooked; of which, molecular repercussions underlying their genetic hazards upon occupational/accidental exposures still remain as an intricate issue and are hitherto unknown. To assess the genotoxic potential of methyl isocyanate in cultured mammalian cells after in vitro exposure, we performed a study in three different normal cell lines MM55.K (mouse kidney epithelial), B/CMBA.Ov (mouse ovarian epithelial), and NIH/3T3 (primary mouse embryonic fibroblast). Cellular DNA damage response was studied for qualitative phosphorylation states of ATM, gammaH2AX proteins and quantitative state of p53 phosphorylation; DNA cell cycle analysis and measure of cellular apoptotic index before and after treatment were also investigated. Our results demonstrate that methyl isocyanate by negatively regulating the DNA damage response pathway, might promote cell cycle arrest, and apoptosis in cultured mammalian cells suggestive of causing genetic alterations. We anticipate that these data along with other studies reported in the literature would help to design better approaches in risk assessment of occupational and accidental exposure to isocyanates. We also predict that increasing knowledge on DNA damage-triggered signaling leading to cell death could provide new strategies for investigating the effects of DNA repair disorders and decreased repair capacity on the toxicity and carcinogenic properties of environmental toxins.

Background: According to the current paradigm, metastases arise from cells of the primary tumor that have intravasated into circulation and carried to distant organs where they extravasate, grow and form new tumors. However, mechanisms underlying the many steps involved in the metastatic cascade remain unexplained. The landmark study by Dr. Isaiah Fidler established that 99% of tumor cells injected IV into mice die within 24 hours of reaching target sites (Fidler, JNCI, 1970). We hypothesized that circulating tumor cells (CTCs) also die upon reaching target organs and release oncogenic cell-free chromatin (cfCh) particles which integrate into their cellular genomes and transform them to form new tumors which masquerade as metastases. Animal Ethics: The study was approved by Institutional Animal Ethics Committee. Experiments and Results: 1) MDA-MB-231 human breast cancer and A375 human malignant melanoma cells were dually labelled in their DNA with BrdU and in their histones with CellLight® Histone 2B GFP and injected IV into mice. Immuno-fluorescence (IF) microscopy of lung tissues excised at 48 hours revealed abundant dually labelled cfCh particles in nuclei of lung cells; 2) Unlabeled MDA-MB-231 and A375 cells were similarly injected, and IF analysis of lung tissues excised at 72 hours revealed 5 - 20 fold increase in all ten hallmarks of cancer defined by Hanahan and Weinberg when compared with un-injected control lung tissues; 3) Mice injected with unlabeled cells were followed for 2-3 months until they developed lung metastasis which were excised and found to contain both mouse and human DNA when examined by highly specific FISH probes. The tumor cells also contained human specific HLA ABC Class I and mouse specific MHC Class II proteins when examined by highly specific mAbs; 4) Metaphase spreads prepared from primary cultures from lung metastasis were found to contain chimeric chromosomes with both mouse and human DNA. The cells expressed both human and mouse specific proteins in the same cells; 5) Several single cell clones developed from primary cultures were also found to contain chimeric chromosomes of mouse and human DNA and to express both mouse and human specific proteins; 6) cfCh particles were isolated from Adriamycin treated dying MDA-MB-231 cells by modification of a method described by us (Mittra et al J Biosciences 2015). When the isolated cfCh particles were injected IV into mice, they went on to develop lung metastasis after ˜ 7 months, thereby directly implicating cfCh in development of metastases. The cfCh induced tumors were again found to contain both mouse and human specific DNA and proteins. Conclusion: Metastases arise as new cancers from cells of target organs transformed by cfCh particles released from dying CTCs. This conclusion challenges the dogma that metastases arise from cells that are derived from the primary tumor. Citation Format: Indraneel Mittra, Venkata Raghuram Gorantla, Soniya Shende, Naveen Kumar Khare, Harshada Wani, Ratnam Prasad, Kavita Pal. Metastases arise as new cancers from cells of target organs transformed by cell-free chromatin particles released from dying circulating tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2875.