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Monaco et al. J Environ Expo Assess 2024;3:18 https://dx.doi.org/10.20517/jeea.2024.10 Page 13 of 18

shift resulted in an increased abundance of Bacillota and Actinobacteria, accompanied by a decrease in
Bacteroidota and Spirochaetota. Consistent with our findings, research involving 4-week-old female ICR
[58]
mice administered 500 or 1,500 mg DEHP/kg BW/day , and pubertal Sprague-Dawley rats dosed with
[45]
3,000 mg DEHP/kg/BW/day reported an increase in the ratio between Bacillota and Bacteroidota in fecal
and cecal contents, respectively. The latter study found that DEHP doses of 300 and 1,000 mg/kg BW/day
did not induce the same microbiota shift . Differences in outcomes can be attributed to the use of different
[45]
animal models and also, the dosage used, duration of exposure, and associated microbial changes .
[45]
To provide additional insight into the alterations in the microbiota, we performed differential analyses at
the genus level. Animals exposed to any DEHP dose had a higher abundance of the Christensenellaceae R-7
and Romboutsia genera in the AC than CON. In the same animals, the abundance of Lachnospiraceae UCG-
004 genus in DEPH20 and DEPH200 piglets was reduced compared to CON. Moreover, the AC contents of
piglets exposed to DEHP200 exhibited lower abundances of Odoribacter and Sphaerochaeta compared to
CON piglets. Interestingly, no difference was observed in either of these genera at the 20 mg DEHP/kg/day
dose.

Christensenellaceae presence in the pig intestine is relatively low, with the colon harboring more of this
bacterium than the ileum and cecum . In humans, Christensenellaceae comprises approximately 0.01% of
[59]
[60]
the fecal microbiota, and its relative abundance is affected by ethnicity, sex, age, and host genotype .
Christensenellaceae has emerged as an important modulator of health, and it is negatively associated with
BMI, fat mass, serum lipids, blood pressure, and impaired glucose metabolism [60,61] . Furthermore, an
increased abundance of species of the family Christensenellaceae has been strongly associated with asthma
and asthma-associated intestinal metabolites in 3-year-old children . However, no other study reported an
[62]
increase in Christensenellaceae after exposure to phthalates. Romboutsia belongs to the Bacillota phylum. Its
prevalence is elevated in various health conditions, such as neurodevelopmental disorders, immune-related
[63]
chronic intestinal inflammation, and gastric cancer . The higher abundance of Romboutsia observed at
either DEHP dose in our study has also been reported in rodent studies [58,64,65] .

Conversely, DEHP reduced the abundance of the genera Lachnospiraceae UCG-004 (Bacillota), Odoribacter
(Bacillota), and Sphaerochaeta (Spirochaeota), all of which are known to metabolize polysaccharides and
non-digestible fiber to produce volatile fatty acids (VFA) [66,67] . VFA are essential for gut integrity, provide an
[68]
energy source for intestinal epithelial cells, and modulate the immune system . In addition, VFA are
important neuro-immuno-endocrine regulators and play significant roles in microbiota-gut-brain crosstalk
[69]
like motility, secretion, and blood flow . Fecal VFA were not measured in this study, but others reported
that administration of high levels of DEHP significantly reduced fecal acetate and butyrate concentrations in
[45]
rats . The authors postulated that the DEHP-dependent change in the microbiota composition modulated
the physiological effects of DEHP (severe organ damage, Th1 inflammatory response, and low fecal
[45]
butyrate). However, these responses are rodent strain- and species-specific .
In the AC, the abundance of VFA-producing bacteria from the genera Holdomanella and Clostridia vadin
BB60 group were lower in DEHP20 than CON, with no difference at the higher dose. A higher abundance
of Holdemanella biformis was inversely correlated with irritable bowel symptoms following fecal microbial
transplant . Additionally, the AC contents of DEHP20 were enriched with Colidextribacter, a genus
[70]
[71]
negatively correlated with BMI z-scores in 5-year-old children who had been born prematurely . Others
have reported that the effects of DEHP on the microbiota are not dose-dependent in rodents [45,58] . Herein,
the overall microbiota structure in the AC and RC clustered more closely to CON than the DEHP200. We
speculate that higher doses are needed to observe significant overall changes in the piglet model.