Oral cancer cell to endothelial cell communication via exosomal miR-21/RMND5A pathway.

Required for meiotic nuclear division 5 homolog A (RMND5A), a novel ubiquitin E3 Ligase, has been reported to correlate with poor prognosis of several cancers. However, its role in endothelial cells has not been reported. In this study, overexpression of RMND5A in human umbilical vein endothelial cells (HUVECs) was performed via lentiviral infection, followed by MTT, would healing and tube formation assay as well as signaling analysis. Moreover, crosstalk between HUVECs and oral squamous cell carcinoma (OSCC) cells was investigated by indirect co-culture with condition medium or tumor cell derived exosomes. Our results showed that overexpression of RMND5A reduced the proliferation, migration and tube formation ability of HUVECs by inhibiting the activation of ERK and NF-κB pathway. Interestingly, OSCC cells can inhibit RMND5A expression of endothelial cells via exosomal miR-21. In summary, our present study unveils that OSCC cells can activate endothelial cells via exosomal miR-21/RMND5A pathway to promote angiogenesis, which may provide novel therapeutic targets for the treatment of OSCC.

Mismatch Repair Protein Msh6Tt Is Necessary for Nuclear Division and Gametogenesis in Tetrahymena thermophila.

DNA mismatch repair (MMR) improves replication accuracy by up to three orders of magnitude. The MutS protein in E. coli or its eukaryotic homolog, the MutSα (Msh2-Msh6) complex, recognizes base mismatches and initiates the mismatch repair mechanism. Msh6 is an essential protein for assembling the heterodimeric complex. However, the function of the Msh6 subunit remains elusive. Tetrahymena undergoes multiple DNA replication and nuclear division processes, including mitosis, amitosis, and meiosis. Here, we found that Msh6Tt localized in the macronucleus (MAC) and the micronucleus (MIC) during the vegetative growth stage and starvation. During the conjugation stage, Msh6Tt only localized in MICs and newly developing MACs. MSH6Tt knockout led to aberrant nuclear division during vegetative growth. The MSH6TtKO mutants were resistant to treatment with the DNA alkylating agent methyl methanesulfonate (MMS) compared to wild type cells. MSH6Tt knockout affected micronuclear meiosis and gametogenesis during the conjugation stage. Furthermore, Msh6Tt interacted with Msh2Tt and MMR-independent factors. Downregulation of MSH2Tt expression affected the stability of Msh6Tt. In addition, MSH6Tt knockout led to the upregulated expression of several MSH6Tt homologs at different developmental stages. Msh6Tt is involved in macronuclear amitosis, micronuclear mitosis, micronuclear meiosis, and gametogenesis in Tetrahymena.

Re-evaluation of CBMN test reference values of persons continuously occupationally exposed to low doses of ionizing radiation in Serbia.

When established, cytokinesis-block micronucleus (CBMN) test reference values should be periodically evaluated according to the recommendations of reference documents. The biodosimetry cytogenetic laboratory of the Serbian Institute of Occupational Health established the CBMN test reference range for people occupationally exposed to ionizing radiation in 2016. Since then, new occupationally exposed persons have been subjected to micronucleus testing, resulting in the need for re-evaluation of existing CBMN test values. The examined population comprised 608 occupationally exposed subjects - 201 from the previous laboratory database and 407 newly examined. Comparison of groups based on gender, age and cigarette consumption did not show significant differences, although certain CBMN values differed significantly between the old and new groups. Duration of occupational exposure, gender, age and smoking habit influenced micronuclei frequency in all three analyzed groups, while no relation was found between type of work and micronucleus test parameters. Since the mean values of all tested parameters in the new group of examinees are within previously established reference ranges, existing values can be used in further research.

One-carbon metabolic enzymes are regulated during cell division and make distinct contributions to the metabolome and cell cycle progression in Saccharomyces cerevisiae.

Enzymes of one-carbon (1C) metabolism play pivotal roles in proliferating cells. They are involved in the metabolism of amino acids, nucleotides, and lipids and the supply of all cellular methylations. However, there is limited information about how these enzymes are regulated during cell division and how cell cycle kinetics are affected in several loss-of-function mutants of 1C metabolism. Here, we report that the levels of the S. cerevisiae enzymes Ade17p and Cho2p, involved in the de novo synthesis of purines and phosphatidylcholine (PC), respectively, are cell cycle-regulated. Cells lacking Ade17p, Cho2p, or Shm2p (an enzyme that supplies 1C units from serine) have distinct alterations in size homeostasis and cell cycle kinetics. Loss of Ade17p leads to a specific delay at START, when cells commit to a new round of cell division, while loss of Shm2p has broader effects, reducing growth rate. Furthermore, the inability to synthesize PC de novo in cho2Δ cells delays START and reduces the coherence of nuclear elongation late in the cell cycle. Loss of Cho2p also leads to profound metabolite changes. Besides the expected changes in the lipidome, cho2Δ cells have reduced levels of amino acids, resembling cells shifted to poorer media. These results reveal the different ways that 1C metabolism allocates resources to affect cell proliferation at multiple cell cycle transitions.

Expression of KIF2A, NDC80, CDK1, and CCNB1 in breast cancer patients: Their interaction and linkage with tumor features and prognosis.

BACKGROUND: Kinesin family member 2A (KIF2A), nuclear division cycle 80 (NDC80), cyclin-dependent kinase 1 (CDK1), and cyclin B1 (CCNB1) exhibit a complex interrelation, which promote cancer progression via multiple ways, whereas their interaction and clinical implications in breast cancer are obscure. Hence, this study aimed to evaluate the correlation among KIF2A, NDC80, CDK1, CCNB1, and their linkage with clinicopathological features and prognosis in breast cancer patients. METHODS: 195 breast cancer patients underwent surgical resection were analyzed. KIF2A, NDC80, CDK1, and CCNB1 expressions were determined by immunohistochemical (IHC) assay and scored by a semiquantitative IHC score or positive cell percentage. RESULTS: KIF2A expression positively associated with NDC80, CDK1, and CCNB1 expressions (all p < 0.01). In terms of tumor features: KIF2A high expression linked with increased T stage (p = 0.011), N stage (p = 0.014), and TNM stage (p = 0.009) but not tumor differentiation (p = 0.651). NDC80 high expression only related to higher N stage (p = 0.010); CDK1 high expression only connected with elevated N stage (p = 0.035) and TNM stage (p = 0.023). In aspect of prognosis, high expression of KIF2A was correlated with worse disease-free survival (DFS) (p = 0.031), while NDC80 high (p = 0.329), CDK1 high (p = 0.276), and CCNB1 positive (p = 0.063) expressions only showed trends to link with poor DFS (without statistical significance). Furthermore, high expression of KIF2A (p = 0.063), NDC80 (p = 0.939), CDK1 (p = 0.413) and positive expression of CCNB1 (p = 0.296) did not relate to overall survival. CONCLUSION: KIF2A correlates with NDC80, CDK1, CCNB1, and may link with advanced tumor stages and poor prognosis in breast cancer patients.

Phytochemical analysis and comprehensive evaluation of pharmacological potential of Artemisia brevifolia Wall. ex DC.

Multitude of diseases and side effects from conventional drugs have surged the use of herbal remedies. Thus, the current study aimed to appraise various pharmacological attributes of Artemisia brevifolia Wall. ex DC. Extracts prepared by successive solvent extraction were subjected to phytochemical and multimode antioxidant assays. Various polyphenolics and artemisinin derivatives were detected and quantified using RP-HPLC analysis. Compounds present in methanol (M) and distilled water (DW) extracts were identified using high resolution mass spectrometry (HRMS). Extracts were pharmacologically evaluated for their antibacterial, antifungal, antimalarial, antileishmanial and antidiabetic potentials. Moreover, cytotoxicity against Artemiasalina, human cancer cell lines and isolated lymphocytes was assessed. Genotoxicity was evaluated using comet, micronucleus and chromosomal aberration assays. Lastly, anti-inflammatory potential was determined through a series of in vitro and in vivo assays using BALB/c mice. Maximum extract recovery (5.95% w/w) was obtained by DW extract. Highest phenolics and flavonoids content, total antioxidant capacity, total reduction potential, percentfree radical scavenging, ß-carotene scavenging and iron chelating activities were exhibited by M extract. RP-HPLC analysis revealed significant amounts of various polyphenolic compounds (vanillic acid, syringic acid, emodin and luteolin), artemisinin, dihydro artemisinin, artesunate and artemether in ethyl acetate (EA) extract. Total 40 compounds were detected through HRMS. A noteworthy antimicrobial activity (MIC 22.22 µg/ml) was exhibited by EA extract against A. fumigatus and several bacterial strains. Maximum antimalarial, antileishmanial, brine shrimp lethality and cytotoxic potential against cancer cells was manifested by EA extract. None of the extracts exhibited genotoxicity and toxicity against isolated lymphocytes. Highest α-amylase and α-glucosidase inhibition capacities were demonstrated by DW extract. Various in-vivo anti-inflammatory models revealed significant (p < 0.05) anti-inflammatory potential of M and DW extracts. In conclusion, present findings divulged theremarkable pharmacological potential of A. brevifolia and endorse its richness in artemisinin.

Trafficking and/or division: Distinct roles of nucleoporins based on their location within the nuclear pore complex.

The nuclear pore complex (NPC) facilitates the trafficking of proteins and RNA between the nucleus and cytoplasm. The role of nucleoporins (Nups) in transport in the context of the NPC is well established, yet their function in tRNA export has not been fully explored. We selected several nucleoporins from different parts of the NPC to investigate their potential role in tRNA trafficking in Trypanosoma brucei. We show that while all of the nucleoporins studied are essential for cell viability, only TbNup62 and TbNup53a function in tRNA export. In contrast to homologs in yeast TbNup144 and TbNup158, which are part of the inner and outer ring of the NPC, have no role in nuclear tRNA trafficking. Instead, TbNup144 plays a critical role in nuclear division, highlighting the role of nucleoporins beyond nucleocytoplasmic transport. These results suggest that the location of nucleoporins within the NPC is crucial to maintaining various cellular processes.

Kinesin Motors in the Filamentous Basidiomycetes in Light of the Schizophyllum commune Genome.

Kinesins are essential motor molecules of the microtubule cytoskeleton. All eukaryotic organisms have several genes encoding kinesin proteins, which are necessary for various cell biological functions. During the vegetative growth of filamentous basidiomycetes, the apical cells of long leading hyphae have microtubules extending toward the tip. The reciprocal exchange and migration of nuclei between haploid hyphae at mating is also dependent on cytoskeletal structures, including the microtubules and their motor molecules. In dikaryotic hyphae, resulting from a compatible mating, the nuclear location, synchronous nuclear division, and extensive nuclear separation at telophase are microtubule-dependent processes that involve unidentified molecular motors. The genome of Schizophyllum commune is analyzed as an example of a species belonging to the Basidiomycota subclass, Agaricomycetes. In this subclass, the investigation of cell biology is restricted to a few species. Instead, the whole genome sequences of several species are now available. The analyses of the mating type genes and the genes necessary for fruiting body formation or wood degrading enzymes in several genomes of Agaricomycetes have shown that they are controlled by comparable systems. This supports the idea that the genes regulating the cell biological process in a model fungus, such as the genes encoding kinesin motor molecules, are also functional in other filamentous Agaricomycetes.

Assaying Cyclin-Dependent Kinase Activity in Synchronized Algal Cultures.

Cyclin-dependent kinases (CDKs) are key regulators of the cell cycle in eukaryotes. Assessing their activity is one of the basic methods used to analyze their function. This is particularly true in synchronized cultures of unicellular organisms, where the entire culture is in the same physiological state. In this chapter, I describe a simple biochemical method to assess CDK activity in algae. Although the results are easier to interpret in the context of synchronized cultures, the method is not limited to them. The protocol requires only standard laboratory equipment and access to a radioactivity working room. The method is applicable to any algal species, including newly developed ones, as it does not require any specific tools. The method can, therefore, be used to widen the portfolio of cell cycle regulatory models within algae.

Nuclear size and shape control.

The nucleus displays a wide range of sizes and shapes in different species and cell types, yet its size scaling and many of the key structural constituents that determine its shape are highly conserved. In this review, we discuss the cellular properties and processes that contribute to nuclear size and shape control, drawing examples from across eukaryotes and highlighting conserved themes and pathways. We then outline physiological roles that have been uncovered for specific nuclear morphologies and disease pathologies associated with aberrant nuclear morphology. We argue that a comparative approach, assessing and integrating observations from different systems, will be a powerful way to help us address the open questions surrounding functional roles of nuclear size and shape in cell physiology.

[CDK1, CCNB1 and NDC80 are associated with prognosis and progression of hepatitis B virus-associated hepatocellular carcinoma: a bioinformatic analysis].

OBJECTIVE: To identify the key genes involved in the transformation of hepatitis B virus (HBV) into hepatocellular carcinoma (HCC) and explore the underlying molecular mechanisms. METHODS: We analyzed the mRNA microarray data of 119 HBV-related HCC tissues and 252 HBV-related non-tumor tissues in GSE55092, GSE84044 and GSE121248 from the GEO database, and the "sva" R package was used to remove the batch effects. Integration analysis was performed to identify the differentially expressed genes (DEGs) in HBV-related liver cancer and liver tissues with HBV infection. The significant DEGs were functionally annotated using GO and KEGG analyses, and the most important modules and hub genes were explored with STRING analysis. Kaplan-Meier and Oncomine databases were used to verify the HCC gene expression data in the TCGA database to explore the correlations of the hub genes with the occurrence, progression and prognosis of HCC. We also examined the expressions of the hub genes in 17 pairs of surgical specimens of HCC and adjacent tissues using RT-qPCR. RESULTS: We identified a total of 121 DEGs and 3 genetic markers in HCC (P < 0.01). These DEGs included cyclin1 (CDK1), cyclin B1 (CCNB1), and nuclear division cycle 80 (NDC80), which participated in cell cycle, pyrimidine metabolism and DNA replication and were highly correlated (P < 0.05). Analysis of the UALCAN database confirmed high expressions of these 3 genes in HCC tissues, which were correlated with a low survival rate of the patients, as shown by Kaplan-Meier analysis of the prognostic data from the UALCAN database. CDK1, CCNB1 and NDC80 were all correlated with the clinical grading of HCC (P < 0.05). The results of RT-qPCR on the surgical specimens verified significantly higher expressions of CDK1, CCNB1 and NDC80 mRNA in HCC tissues than in the adjacent tissues. CONCLUSION: CDK1, CCNB1 and NDC80 genes can be used as prognostic markers of HBV-related HCC and may serve as potential targets in preclinical studies and clinical treatment of HCC.

Expansion Microscopy Reveals Plasmodium falciparum Blood-Stage Parasites Undergo Anaphase with A Chromatin Bridge in the Absence of Mini-Chromosome Maintenance Complex Binding Protein.

The malaria parasite Plasmodium falciparum undergoes closed mitosis, which occurs within an intact nuclear envelope, and differs significantly from its human host. Mitosis is underpinned by the dynamics of microtubules and the nuclear envelope. To date, our ability to study P. falciparum mitosis by microscopy has been hindered by the small size of the P. falciparum nuclei. Ultrastructure expansion microscopy (U-ExM) has recently been developed for P. falciparum, allowing the visualization of mitosis at the individual nucleus level. Using U-ExM, three intranuclear microtubule structures are observed: hemispindles, mitotic spindles, and interpolar spindles. A previous study demonstrated that the mini-chromosome maintenance complex binding-protein (MCMBP) depletion caused abnormal nuclear morphology and microtubule defects. To investigate the role of microtubules following MCMBP depletion and study the nuclear envelope in these parasites, we developed the first nuclear stain enabled by U-ExM in P. falciparum. MCMBP-deficient parasites show aberrant hemispindles and mitotic spindles. Moreover, anaphase chromatin bridges and individual nuclei containing multiple microtubule structures were observed following MCMBP knockdown. Collectively, this study refines our understanding of MCMBP-deficient parasites and highlights the utility of U-ExM coupled with a nuclear envelope stain for studying mitosis in P. falciparum.

Nuclear positioning during development: Pushing, pulling and flowing.

The positioning of the nucleus, the central organelle of the cell, is an active and regulated process crucially linked to cell cycle, differentiation, migration, and polarity. Alterations in positioning have been correlated with cell and tissue function deficiency and genetic or chemical manipulation of nuclear position is embryonic lethal. Nuclear positioning is a precursor for symmetric or asymmetric cell division which is accompanied by fate determination of the daughter cells. Nuclear positioning also plays a key role during early embryonic developmental stages in insects, such as Drosophila, where hundreds of nuclei divide without cytokinesis and are distributed within the large syncytial embryo at roughly regular spacing. While the cytoskeletal elements and the linker proteins to the nucleus are fairly well characterised, including some of the force generating elements driving nuclear movement, there is considerable uncertainty about the biophysical mechanism of nuclear positioning, while the field is debating different force models. In this review, we highlight the current body of knowledge, discuss cell context dependent models of nuclear positioning, and outline open questions.

Non-ribosomal insights into ribosomal P2 protein in Plasmodium falciparum-infected erythrocytes.

The enormous complexity of the eukaryotic ribosome has been a real challenge in unlocking the mechanistic aspects of its amazing molecular function during mRNA translation and many non-canonical activities of ribosomal proteins in eukaryotic cells. While exploring the uncanny nature of ribosomal P proteins in malaria parasites Plasmodium falciparum, the 60S stalk ribosomal P2 protein has been shown to get exported to the infected erythrocyte (IE) surface as an SDS-resistant oligomer during the early to the mid-trophozoite stage. Inhibiting IE surface P2 either by monoclonal antibody or through genetic knockdown resulted in nuclear division arrest of the parasite. This strange and serendipitous finding has led us to explore more about un-canonical cell biology and the structural involvement of P2 protein in Plasmodium in the search for a novel biochemical role during parasite propagation in the human host.

The histone chaperone Nrp1 is required for chromatin stability and nuclear division in Tetrahymena thermophila.

Histone chaperones facilitate DNA replication and repair by promoting chromatin assembly, disassembly and histone exchange. Following histones synthesis and nucleosome assembly, the histones undergo posttranslational modification by different enzymes and are deposited onto chromatins by various histone chaperones. In Tetrahymena thermophila, histones from macronucleus (MAC) and micronucleus (MIC) have been comprehensively investigated, but the function of histone chaperones remains unclear. Histone chaperone Nrp1 in Tetrahymena contains four conserved tetratricopepeptide repeat (TPR) domains and one C-terminal nuclear localization signal. TPR2 is typically interrupted by a large acidic motif. Immunofluorescence staining showed that Nrp1 is located in the MAC and MICs, but disappeared in the apoptotic parental MAC and the degraded MICs during the conjugation stage. Nrp1 was also colocalized with α-tubulin around the spindle structure. NRP1 knockdown inhibited cellular proliferation and led to the loss of chromosome, abnormal macronuclear amitosis, and disorganized micronuclear mitosis during the vegetative growth stage. During sexual developmental stage, the gametic nuclei failed to be selected and abnormally degraded in NRP1 knockdown mutants. Affinity purification combined with mass spectrometry analysis indicated that Nrp1 is co-purified with core histones, heat shock proteins, histone chaperones, and DNA damage repair proteins. The physical direct interaction of Nrp1 and Asf1 was also confirmed by pull-down analysis in vitro. The results show that histone chaperone Nrp1 is involved in micronuclear mitosis and macronuclear amitosis in the vegetative growth stage and maintains gametic nuclei formation during the sexual developmental stage. Nrp1 is required for chromatin stability and nuclear division in Tetrahymena thermophila.

Asymmetric nuclear division in neural stem cells generates sibling nuclei that differ in size, envelope composition, and chromatin organization.

Although nuclei are the defining features of eukaryotes, we still do not fully understand how the nuclear compartment is duplicated and partitioned during division. This is especially the case for organisms that do not completely disassemble their nuclear envelope upon entry into mitosis. In studying this process in Drosophila neural stem cells, which undergo asymmetric divisions, we find that the nuclear compartment boundary persists during mitosis thanks to the maintenance of a supporting nuclear lamina. This mitotic nuclear envelope is then asymmetrically remodeled and partitioned to give rise to two daughter nuclei that differ in envelope composition and exhibit a >30-fold difference in volume. The striking difference in nuclear size was found to depend on two consecutive processes: asymmetric nuclear envelope resealing at mitotic exit at sites defined by the central spindle, and differential nuclear growth that appears to depend on the available local reservoir of ER/nuclear membranes, which is asymmetrically partitioned between the two daughter cells. Importantly, these asymmetries in size and composition of the daughter nuclei, and the associated asymmetries in chromatin organization, all become apparent long before the cortical release and the nuclear import of cell fates determinants. Thus, asymmetric nuclear remodeling during stem cell divisions may contribute to the generation of cellular diversity by initiating distinct transcriptional programs in sibling nuclei that contribute to later changes in daughter cell identity and fate.

Cytogenetic and oxidative effects of three lichen extracts on human peripheral lymphocytes.

In the present study, we investigated cytogenetic and oxidative [total antioxidant capacity (TAC), total oxidant status (TOS)] effects of methanol and water extracts of Cladonia chlorophaea (Flörke ex Sommerf.) Sprengel, Dermatocarpon miniatum (L.) W.Mann and Parmelia saxatilis (L.) Ach. on cultured human lymphocytes. In addition, different phenolic compounds in the extracts were quantified by high performance liquid chromatography (HPLC) analysis. As a result of HPLC analysis, methanol extracts of all lichen species tested had higher phenolic compounds. Likewise, methanol extracts of each lichen increased TAC levels in lymphocytes more than water extracts. The TOS levels of the cells treated with different concentrations (1-100 mg/L) of the extracts decreased due to the increasing concentration of the extracts. Genotoxicity experiments revealed that the tested lichen extracts did not significantly increase (p > 0.05) the level of genotoxicity on human peripheral lymphocyte culture compared to the negative control group. The results showed that C. chlorophaea, D. miniatum and P. saxatilis lichens, which were found to be a rich source of phenolic compounds, might be of interest in the pharmaceutical and food industries.

An update on cell division of Giardia duodenalis trophozoites.

Giardia duodenalis is a flagellated protozoan that is responsible for many cases of diarrheal disease worldwide and is characterized by its great divergence from the model organisms commonly used in studies of basic cellular processes. The life cycle of Giardia involves an infectious cyst form and a proliferative and mobile trophozoite form. Each Giardia trophozoite has two nuclei and a complex microtubule cytoskeleton that consists of eight flagellar axonemes, basal bodies, the adhesive disc, the funis and the median body. Since the success of Giardia infecting other organisms depends on its ability to divide and proliferate efficiently, Giardia must coordinate its cell division to ensure the duplication and partitioning of both nuclei and the multiple cytoskeletal structures. The purpose of this review is to summarize current knowledge about cell division and its regulation in this protist.

miR-590-5p targets RMND5A and promotes migration in pancreatic adenocarcinoma cell lines.

Required for meiotic nuclear division 5 homolog A (RMND5A) functions as an E3 ubiquitin ligase. To date, few studies have investigated the role of RMND5A in cancer. In the present study, the expression levels of RMND5A in multiple types of cancer were analyzed using the Gene Expression Profiling Interactive Analysis platform. The results revealed that RMND5A was highly expressed and associated with overall survival in patients with pancreatic adenocarcinoma (PAAD). A wound-healing assay revealed that RMND5A overexpression significantly increased cell migration in the PAAD cell lines AsPC-1 and PANC-1. In silico analysis predicted that RMND5A was a potential target of microRNA(miR)-590-5p. Further in vitro experiments demonstrated that overexpression of miR-590-5p downregulated the expression levels of RMND5A and decreased the migratory ability of the AsPC-1 and PANC-1 cell lines. In addition, overexpression of miR-590-5p attenuated the promoting effects of RMND5A on the migration of AsPC-1 and PANC-1 cells. The results of the present study may further elucidate the mechanisms underlying PAAD progression and provide novel targets for the treatment of PAAD.

Consumption pattern and genotoxic potential of various smokeless tobacco products in Assam, India: A public health concern.

Smokeless tobacco (SLT) consumption is presumed to be one of the major causes of high incidence of oral cancer in India. The present study aimed to document various types of SLT products consumed and their potential impact on the genome instability on the population from Assam state in Northeast India. A cross-sectional study (n = 5000) showed that 60.56 % of the study population consumed at least one of the three forms (sadagura, zarda and khaini) of SLT of which 52.0 % were only sadagura users. Genotoxicity assessment using buccal cytome assay in 240 age and sex matched volunteers revealed that except for zarda, other forms of SLT induced significantly higher incidence micronuclei in the buccal epithelial cells compared to the control individuals. Similar effects were also observed in other cytome parameters related to cell proliferation, cytokinesis defects and cell death. Significantly higher incidence of micronucleus was observed among sadagura and khaini users in lymphocyte cytokinesis-blocked micronucleus assay. The addition of lime in sadagura increased the pH and anion levels which possibly result in higher absorption and may lead to the development of cellular anomalies.