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In vitro study of chlorine dioxide on porcine intestinal epithelial cell gene markers

Background Chlorine dioxide (ClO2) is an inorganic, potent biocide and is available in highly purified aqueous solution. It can be administered as an oral antiseptic in this form. Objectives Our aim is to determine the level of inflammatory markers and cytochrome genes expressed by enterocytes exposed to different concentrations of hyperpure chlorine dioxide solution. Methods Porcine jejunal enterocyte cell (IPEC‐J2) cultures were treated with the aqueous solution of hyper‐pure chlorine dioxide of various concentrations. We determined the alterations in mRNA levels of inflammatory mediators, such as IL6, CXCL8/IL8, TNF, HSPA6 (Hsp70), CAT and PTGS2 (COX2); furthermore, the expression of three cytochrome genes (CYP1A1, CYP1A2, CYP3A29) were analysed by quantitative PCR method. Results The highest applied ClO2 concentration reduced the expression of all three investigated CYP genes. The gene expression of PTGS2 and CAT were not altered by most concentrations of ClO2. The expression of IL8 gene was reduced by all applied concentrations of ClO2. TNF mRNA level was also decreased by most ClO2 concentrations used. Conclusions Different concentrations of chlorine dioxide exhibited immunomodulatory activity and caused altered transcription of CYP450 genes in porcine enterocytes. Further studies are needed to determine the appropriate ClO2 concentration for oral use in animals.

Due to widespread and continuously emerging antimicrobial resistance, antimicrobial drug consumption is required to be significantly reduced in animal husbandry. Among arising alternative solutions, the hyperpure ClO 2 (patent: Noszticzius et al., 2007) could be an ideal biocidal additive to the diet of food-producing animals. It is a great advantage that microbial resistance to chlorine dioxide is unlikely because it acts on the thiol group which is fundamental in all living organisms (Noszticzius et al., 2013). Furthermore, during this pandemic era, the use of safe antiseptics is gaining prominence, as their use has also become generic in everyday life.
Chlorine dioxide has found to be highly biocidal in low concentrations on intestinal biota while simultaneously at this same concentration having no negative effect to daily weight gain (Akamatsu et al., 2012). It is proposed that the animals can safely drink it, without any adverse effect (Ma et al., 2017).
There are no scientific data regarding the biological effects of chlorine dioxide as an intestinal antiseptic in swine. We have chosen the porcine intestinal epithelial cell line (IPEC-J2) to begin exploring these effects. The gene expression profile of IPEC-J2 cell cultures makes them suitable for studying the effects of added compounds (Rhoads et al., 1997, Arce et al., 2010, Vergauwen et al., 2015, Razzuoli et al., 2018. The IPEC-J2 cells are non-cancerous intestinal columnar epithelial cells that were isolated from neonatal piglet mid-jejunum (Langerholc et al., 2011). According to these aspects, IPEC-J2 cell line is an adequate model for preliminary studies investigating the effect of chlorine dioxide.
Our aim is to determine whether chlorine dioxide has any effect on inflammatory markers and cytochrome genes expressed by the small intestinal epithelia. The cytochrome P450 (CYP450) enzymes are involved in drug metabolism, by investigating them we might gain information about the drug interaction properties of this biocidal agent.

Cell line and culture conditions
The non-transformed porcine intestinal epithelial cell line IPEC-J2, originally isolated from jejunal epithelia of a neonatal unsuckled piglet (Schierack et al., 2006), was a kind gift of Dr. Jody Gookin, Department IPEC-J2 cells were seeded onto six-well plates (Corning Inc., Corning, NY, USA), coated with 8 μg/cm 2 rat tail collagen type I (Sigma-Aldrich, Steinheim, Germany), at a density of 10 6 cells/ml; the volume of complete medium was 2.5 ml. Cells could adhere for 24 h before being washed and re-fed every other day.

Cell viability test
Influence of chlorine dioxide on the viability of enterocytes was tested. A twofold serial dilution of hyperpure chlorine dioxide solution (Solvocid ® Vet, Solumium Ltd, Hungary) was prepared across 7 points in phosphate-buffered saline (PBS) from 300 to 4.7 ppm. IPEC-J2 cells were seeded onto a 96-well plate and incubated with the test substances for 15 min at 37 • C in 5% CO 2 . After treatment, the cells were washed two times with PBS and were placed back to the thermostat in complete medium. Viability of IPEC-J2 cells was measured 24 h after treatment by Neutral red uptake assay as described by Repetto et al. (2008).

Quantitative PCR measurements
One hour after the treatment, culture medium was removed, cells were collected, mRNA was extracted and cDNA was synthetised according to Palócz et al. (2016).

Statistical analyses
Relative

Effect of chlorine dioxide on relative expression of inflammatory genes
The gene expression of PTGS2 and CAT were not altered by most con-

F I G U R E 2
The relative gene expressions of CAT and PTGS2/COX2 at various ClO 2 concentrations from 0.07 to 2.22 mM in porcine jejunal cell cultures. Results are expressed as mean mRNA expression ratio relative to controls (n = 6/group). Significant differences are shown in comparison to untreated controls (*p < 0.05, **p < 0.01). Data are shown as means ± SD. CAT, catalase; PTGS2, prostaglandin-endoperoxide synthase 2, also known as cyclooxygenase 2 (COX2)

F I G U R E 3
The relative gene expressions of IL6, CXCL8/IL8 and TNF at various ClO 2 concentrations from 0.07 to 2.22 mM in porcine jejunal cell cultures. Results are expressed as mean mRNA expression ratio relative to controls (n = 6/group). Significant differences are shown in comparison to untreated controls (*p < 0.05, **p < 0.01). Data are shown as means ± SD. CXCL8, C-X-C motif chemokine ligand 8; IL, interleukin; TNF, tumour necrosis factor and 1.11 mM (150 and 75 ppm) -increased the gene expression, and the lower concentrations -0.28, 0.14 and 0.07 mM (18.75, 9.38, and 4.69 ppm, respectively) -decreased the gene expression (Figure 4).

Effect of chlorine dioxide on relative expression of CYP450 genes
The highest ClO 2 concentration -2.22 mM (150 ppm Results are expressed as mean mRNA expression ratio relative to controls (n = 6/group). Significant differences are shown in comparison to untreated controls (* and # p < 0.05, **p < 0.01). Data are shown as means ± SD. HSPA6, heat shock protein family A (Hsp70) member 6 F I G U R E 5 The relative gene expressions of CYP1A1, CYP1A2 and CYP3A29 at various ClO 2 concentrations from 0.07 to 2.22 mM in porcine jejunal cell cultures. Results are expressed as mean mRNA expression ratio relative to controls (n = 6/group). Significant differences are shown in comparison to untreated controls (# p < 0.05, ** and ## p < 0.01). Data are shown as means ± SD. CYP, cytochrome P450 after incubation with the lower concentrations of ClO 2 : 0.07-0.56 mM (4.69-37.5 ppm).

DISCUSSION
Based on the mechanism of action of chlorine dioxide, we hypothesise that negligible amount would reach the small intestine when administered via feed. However, via drinking water administration a significant portion of the administered amount could pass through the stomach if empty, and the incidence of this is increased by long-term use or inadequate inclusion ratio.
The examined inflammatory and cytochrome genes expressed by the non-cancerous intestinal epithelial cells were influenced by certain concentrations of chlorine dioxide. The lower concentrations applied decreased the gene expression of HSPA6. Heat shock protein 70 is crucial in cell survival: it can prevent apoptosis, repair damaged proteins (Murphy, 2013) and reduce mitochondrial and cellular ROS production (Li et al., 2018).
TNF induces apoptosis (Guicciardi et al., 2000) and also damages the epithelial barrier integrity and stimulate the inflammatory process, inhibition of TNF decreases the production of proinflammatory mediators, reduces the proapoptotic markers and the ileal paracellular permeability (Halpern et al., 2006). Overall, downregulation of TNF can hinder acute or chronic inflammation and tissue necrosis.
IL6 is an acute phase immune mediator that cooperates with host defence when infections or injuries occur. However, permanent presence of IL6 leads to chronic inflammation and the development of other immune-mediated diseases (Tanaka & Kishimoto, 2014). IL8/CXCL8 is The 0.28 mM ClO 2 concentration decreased the CAT mRNA level.
Inhibition of catalase will directly result in increased production of reactive oxygen species, which consequently leads to higher apoptosis rate (Majumder et al., 2017).
Concerning drug metabolism, the 1.11 mM ClO 2 concentration exerted no effect on the transcription of the three investigated CYP genes of porcine intestinal cells. The lower ClO 2 concentrations (0.07 and 0.14 mM) resulted in increased expression of the CYP1A1 gene; this six-to eight-time increase might result in elevated protein levels which would lead to altered metabolism of the known CYP1A1 substrate drugs such as azole antifungals (Velík et al., 2004) and quinolone antimicrobials (Li et al., 2018). Finding the recommended oral dose that has no effect on the xenobiotic metabolising enzymes would be crucial to avoid drug-feed interactions or any alteration in drug biotransformation.
There were very few studies investigating the effect of per os chlorine dioxide in food-producing animals; one study demonstrated how 0.4 and 0.5 ppm chlorine dioxide oral treatment for 28 days decreased the occurrence of pathogenic microorganisms such as Escherichia coli and Salmonella in the intestinal tract while not negatively impacting the daily weight gain and feed palatability of broiler chickens (Sultan et al., 2015). In another study, the broiler feed was supplemented with ClO 2 powder at 500 and 1000 ppm for 35 days; consequently, the number of E. coli was reduced in the ileum and cecum (Ahmed et al., 2015).
These results make chlorine dioxide a promising antiseptic agent for oral use. The introduction of the use of chlorine dioxide in pig diets could also be effective in preventing the colonisation and multiplication of pathogens.
According to the outcome of our in vitro studies on the non- was provided by Solumium Ltd.

CONFLICT OF INTEREST
The authors declare no conflict of interest.

ETHICS
The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to. No ethical approval was required as no live animal experiments were performed during this study.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.