Isolation of DiNP-Degrading Microbes from the Mouse Colon and the Influence DiNP Exposure Has on the Microbiota, Intestinal Integrity, and Immune Status of the Colon

Abstract

Di-isononyl phthalate (DiNP) is a plasticizer used to impart flexibility or stability in a variety of products including polyvinyl chloride, cable coatings, artificial leather, and footwear. Previous studies have examined the impact of DiNP on gut integrity and the colonic immune microenvironment, but this study further expands the research by examining whether DiNP exposure alters the colonic microbiota and various immune markers. Previous studies have also revealed that environmental microbes degrade various phthalates, but no studies have examined whether anaerobic gut bacteria can degrade DiNP. Thus, this study tested the hypothesis that DiNP exposure alters the gut microbiota and immune-related factors, and that anaerobic bacteria in the gut can utilize DiNP as the sole carbon source. To test this hypothesis, adult female mice were orally dosed with corn oil or various doses of DiNP for 10–14 consecutive days. After the treatment period, mice were euthanized during diestrus. Colonic contents were collected for full-length 16S rRNA gene sequencing to identify the bacteria in the colon contents. Sanger sequencing of the 16S rRNA gene was used to identify bacteria that were able to grow in Bacteroides minimal media with DiNP as the sole carbon source. Colon tissues were collected for immunohistochemistry of immune(-related) factors. An environmentally relevant dose of DiNP (200 µg/kg) significantly increased a Lachnoclostridium taxon and decreased Blautia compared to the control. Collectively, minimal changes in the colonic microbiota were observed as indicated by non-significant beta-diversities between DiNP treatments and control. Furthermore, three strains of anaerobic bacteria derived from the colon were identified to use DiNP as the sole carbon source. Interestingly, DiNP exposure did not alter protein levels of interleukin-6, tumor necrosis factor alpha, claudin-1, and mucin-1 compared to the control. Collectively, these findings show that DiNP exposure alters the gut microbiota and that the gut contains DiNP-degrading microbes.

Impact of Plasticizer on the Intestinal Epithelial Integrity and Tissue-Repairing Ability within Cells in the Proximity of the Human Gut Microbiome

“…DINP-co-incubated microbial extracts further suppresses anti-inflammatory genes IL10, IL10R2 and IRF4, which may skew the macrophage differentiation balance. It has been reported that a high concentration of plasticizers is required to exert direct effects on expressions of some tight junction proteins and cytokines in quail and mouse [55][56][57]. By contrast, the exact direct or indirect effect(s) of plasticizers on human enterocyte or macrophages have not been studied.…”

The intestinal microbiota as mediators between dietary contaminants and host health

“…DiNP administration over 14 days to adult female mice in occupationally and postnatally relevant doses (20 µg/kg/bw and 200 µg/kg/bw, respectively) led to minimal changes in relative abundance of microbiome species compared with control, with the principal change noted as an increase in the relative abundance in Blautia in the 20 µg/kg/bw/day DiNP group, though this change was also present in the control; the authors noted that the abundance of Blautia was more variable in the control mice, pointing to a more consistent effect of DiNP on relative abundance following exposure in this sample. 99 Importantly, authors used isolated genomic DNA coupled with 16S rRNA gene sequencing and polymerase chain reaction PCR amplification to identify three species capable of utilizing DiNP as a carbon source ( Proteus mirabilis strain ATCC 29,906, Desulfitobacterium hafniense DCB-2 and Paenibacillus barengoltzii strain NBRC 101,215 ). Outside of in vivo studies in rodents, Kolb, O’Loughlin & Gsell (2019) characterized phthalate-degrading bacteria in the microbiomes of two species of Asian carp.…”