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Monaco et al. J Environ Expo Assess 2024;3:18 https://dx.doi.org/10.20517/jeea.2024.10 Page 11 of 18

Table 7. Bacterial genera that significantly differed in the rectal contents of piglets supplemented orally with 0 (CON), 20
(DEHP20), or 200 (DEHP200) mg DEHP/kg BW/day
DEHP20/CON DEHP200/CON DEHP200/DEHP20
Bacterial genus Base mean
LFC Padj LFC Padj LFC Padj
Bacteroidota
Odoribacter 589.2 1.00 0.945 -6.23 0.175 -7.23 0.063
Sanguibacteroides 97.0 -0.39 0.952 -3.84 0.107 -3.45 0.084
Alistipes 676.0 1.64 0.525 -1.48 0.469 -3.12 0.063
Bacillota
[Clostridium] innocuum 5.5 -0.17 0.985 3.01 0.175 3.18 0.077
Holdemanella 589.8 -6.78 0.004 2.80 0.399 9.58 < 0.001
Turicibacter 153.8 -0.67 0.934 1.99 0.322 2.66 0.085
Leuconostoc 3.1 -4.06 0.281 0.59 0.813 4.65 0.085
Lactococcus 6.5 -3.20 0.281 0.60 0.792 3.79 0.077
Streptococcus 218.6 -2.08 0.281 0.51 0.779 2.60 0.063
RF39 240.0 1.28 0.934 -4.09 0.175 -5.36 0.063
Eubacterium 50.6 -0.38 0.945 2.44 0.175 2.82 0.063
[Eubacterium] fissicatena 428.4 -0.77 0.720 -2.11 0.107 -1.35 0.266
Colidextribacter 65.4 2.63 0.135 2.77 0.107 0.14 0.925
Anaerovorax 9.8 0.39 0.945 -2.28 0.175 -2.67 0.063
Family XIII AD3011 56.6 -0.97 0.525 -2.19 0.026 -1.21 0.210

Data were analyzed using DESeq2 package of R, n = 7 to 8 per group. Only the genus with mean relative abundance ≥ 0.01% and present in ≥
20% of the samples were evaluated. Padj, P-value adjusted by the Benjamini–Hochberg method to control false discovery rate; Padj ≤ 0.1 was
considered statistically significant. DEHP: Di-(2-ethylhexyl) phthalate; BW: body weight; base mean: means of normalized counts for all samples;
LFC: log2 fold-change.

The influence of phthalates on RC revealed a significant decrease in the relative abundance of
[Eubacterium] Fissicatena and Anaerovoracaceae Family XIII AD3011 in DEHP200 compared to CON
alone [Table 7]. Meanwhile, the reduction in Sanguibacteroides was relative to both CON and DEHP20.
Holdemanella levels decreased in DEHP20 compared to CON and DEHP200. In the pairwise comparison
between DEHP200 and DEHP20, the genera [Clostridium] innocuum, Turicibacter, Leuconostoc,
Lactococcus, Streptococcus, and Eubacterium were elevated, while Odoribacter, Alistipes, RF29, and
Anaerovorax were reduced.

DISCUSSION
This study aimed to investigate whether early postnatal exposure to DEHP results in deviations in intestinal
development and function, as well as alterations in gut microbiota, compared to no exposure in neonatal
pigs. The DEHP doses were in the range used in studies in rodents [37-39] and swine [40,41] or described to be the
estimated exposure of infants in a neonatal intensive care unit . We observed no effects on whole BW gain
[42]
or organ growth, whereas effects on intestinal villus structure, disaccharidase activity, and colonic
microbiota were observed.

Based on urinary metabolite profiles, neonatal pigs appear to metabolize DEHP similarly to newborn infants
with respiratory distress who received IV fluid treatment . Specifically, MEHHP, MEHP, MEOHP, and
[43]
MECCP concentrations in DEHP-exposed piglets were significantly higher than controls and increased in a
dose-dependent fashion. Diesters of phthalate metabolites are degraded by esterase and lipase to form
[44]
monoesters that may further be transformed into oxidative metabolites in the intestine or other tissues .
Urinary metabolites are used as indicators of phthalates exposure and are used for biomonitoring of