Phthalates Impair Germ Cell Development in the Human Fetal Testis
            <i>in Vitro</i>
            without Change in Testosterone Production

Lambrot, Romain; Muczynski, Vincent; Lécureuil, Charlotte; Angenard, Gaëlle; Coffigny, Hervé; Pairault, Catherine; Moison, Delphine; Frydman, René; Habert, René; Rouiller-Fabre, Virginie

doi:10.1289/ehp.11146

Cited by 168 publications

(181 citation statements)

References 59 publications

(82 reference statements)

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“…Fetal human testis explants exposed to MEHP also failed to exhibit any changes in testosterone production although germ cell loss was observed; Leydig cell aggregation was not evaluated (Lambrot et al 2009). Recently, when fetal human testis or fetal rat testis explants were xenografted into castrate male nude mice and exposed to DBP, serum testosterone was compromised in mice with rat testis grafts but not in mice with human testis grafts (Mitchell et al 2012).…”

Section: Introductionmentioning

confidence: 99%

Novel molecular targets associated with testicular dysgenesis induced by gestational exposure to diethylhexyl phthalate in the rat: a role for estradiol

Klinefelter¹,

Laskey²,

Winnik³

et al. 2012

Reproduction

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Significant research has been focused on phthalate-induced alterations in male reproductive development. Studies on rodents have prompted the notion that a syndrome exists in the human male which includes phenotypic alterations such as hypospadias, cryptorchidism, poor semen quality, and even testicular cancer. Each phenotype in this 'testicular dysgenesis syndrome' is predicated on reduction in testosterone production by the fetal Leydig cell. We sought to examine the relationship between dysgenesis and steroidogenic capacity in the fetal rat testis more stringently by incorporating lower exposures than those typically used, conducting a comprehensive, non-targeted quantitative evaluation of the fetal testis proteome, and relating alterations in individual proteins to the capacity of the fetal Leydig cell to produce testosterone, and histopathology of the fetal testis. Pregnant dams were dosed orally from gestation day (GD) 13-19 with 0, 10, or 100 mg diethylhexyl phthalate (DEHP)/kg body weight per day. Each endpoint was represented by 16 l. Clustering of Leydig cells occurred before any significant decrease in the capacity of the GD19 Leydig cell to produce testosterone. At 100 mg DEHP/kg, testosterone production was reduced significantly, Leydig cell clusters became quite large, and additional dysgenetic changes were observed in the fetal testis. Of 23 proteins whose expression was altered significantly at both DEHP exposure levels, seven were found to be correlated with and predictive of the quantified endpoints. None of these proteins have been previously implicated with DEHP exposure. Notably, pathway analysis revealed that these seven proteins fit a pathway network in which each is regulated directly or indirectly by estradiol.

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Section: Introductionmentioning

confidence: 99%

Novel molecular targets associated with testicular dysgenesis induced by gestational exposure to diethylhexyl phthalate in the rat: a role for estradiol

Klinefelter¹,

Laskey²,

Winnik³

et al. 2012

Reproduction

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“…An alternative method for studying the impact of factors on fetal testicular tissues has been provided by organ cultures (Bendsen et al 2001, Robinson et al 2003, Lambrot et al 2006b, Hallmark et al 2007). Expression of AMH and germ cell mitosis has been reported (Bendsen et al 2001, Lambrot et al 2009). …”

Section: Discussionmentioning

confidence: 99%

“…Previous reports have described studies using organ cultures of human fetal testis recovered during the first (Lambrot et al 2009) and second trimester (Robinson et al 2003, Hallmark et al 2007) to investigate the impact of various agents including retinoic acid and environmental toxicants (Lambrot et al 2006a. Others have reported the use of short-term cultures (up to 6 days) of cells obtained following enzymatic digestion of testes obtained from infants and children to study the impact of hormones such as LH and FSH on gene expression (Berensztein et al 1992, Rivarola et al 1995.…”

Section: Introductionmentioning

confidence: 99%

Establishment of long-term monolayer cultures of somatic cells from human fetal testes and expansion of peritubular myoid cells in the presence of androgen

Cowan¹,

Childs²,

Anderson³

et al. 2010

Reproduction

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The somatic (Sertoli cell (SC), Leydig cell (LC), and peritubular myoid (PTM) cell) cells play key roles in development of the fetal testis. We established monolayer cultures from second trimester human testes and investigated the pattern of expression of cell-lineage characteristic mRNAs. Expression of some SC-associated genes (SRY, SOX9, WT1, GATA4, and SF1) was detectable up to and including passage 3 (P3), while others (anti-Mü llerian hormone; desert hedgehog) present prior to dissociation were not expressed in the cultured cells. Transcripts encoding the androgen receptor were expressed but addition of dihydrotestosterone (DHT) had no impact on expression of mRNAs expressed in SC or LC. Total concentrations of mRNAs encoding smooth muscle actin (ACTA2) and desmin increased from P1 to P3; an increasing proportion of the cells in the cultures were immunopositive for ACTA2 consistent with proliferation/differentiation of PTM cells. In conclusion, somatic cell monolayer cultures were established from human fetal testes; these cultures could form the basis for future studies based on isolation of purified populations of somatic cells and manipulation of gene expression that is difficult to achieve with organ culture systems. Our results suggest that fetal SC do not maintain a fully differentiated phenotype in vitro, yet PTM (ACTA2 positive) cells readily adapt to monolayer culture conditions in the presence of DHT. This culture system provides an opportunity to study the impact of regulatory factors on gene expression in PTM cells, a population thought to play a key role in mediating androgen action within the developing testis.

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“…Entsprechende Experimente liegen für DEHP oder MEHP nicht vor. In Ex-situ-Organkultursystem-Experimenten hatte 0,1 mM MEHP keinen Effekt auf die Testosteronproduktion in fetalen Hoden des Menschen (Lambrot et al 2009) und induzierte nur bei Mäuse-und Rattenhodenkulturen vielkernige Keimzellen, nicht jedoch in denen des Menschen (Habert et al 2014). Aus diesen Experimenten wird geschlossen, dass humane Leydigzellen gegen-über dem antiandrogenen Effekt der Phthalate resistent sind.…”

Section: Humanrelevanzunclassified

Di‐(2‐ethylhexyl)phthalat (DEHP) [MAK Value Documentation in German language, 2016]

Hartwig

Arand²

2016

The MAK‐Collection for Occupational Health and Safety

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The German Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area has re‐evaluated the developmental toxicity of di(2‐ethylhexyl) phthalate (DEHP). In 2014, the maximum concentration at the workplace (MAK value) of DEHP was set to 2 mg/m 3 for the inhalable fraction. In some studies, a correlation between prenatal DEHP exposure and effects on anogenital distance of male newborns, birth weight and pregnancy duration in humans is found; however, the results are not consistent. Teratogenicity is observed in rats only at maternally toxic doses of 1000 mg/kg bw and day and above, in mice at 91 mg/kg bw and day. NOAELs for teratogenicity are 200 mg/kg bw and day for rats and 44 mg/kg bw and day for mice, the latter corresponding to 29 mg/m 3 after scaling to a concentration at the workplace. In contrast to mouse and marmoset, the rat is very sensitive to the effects of DEHP on the male reproductive organs. From a three‐generation study, the Commission deduces a NOAEL for pre‐ and postnatal developmental toxicity to the male reproductive organs and for foetotoxicity of 46 mg/kg bw and day for rats, corresponding to 54 mg/m 3 after scaling to a concentration at the workplace. The Commission does not consider the reported effects – small testes, reduced absolute weights of testis and epididymis – to be adverse because they do not show correlating consequences for the next generation, there is no dose‐response relationship to changes in relative organ weights and histopathological correlates are not observed. Germ cell organization, mRNA expression and protein level of StAR („steroidogenic acute regulatory protein“) in testes are affected after in utero exposure to 100 mg/kg bw and day and above. These effects resulted in a NOAEL for prenatal developmental effects of the male reproductive organs of 30 mg/kg bw and day for rats, corresponding to 35 mg/m 3 . For mice a NOAEL for foetotoxicity as well as prenatal developmental toxicity of 48 mg/kg bw and day is derived in prenatal developmental studies, corresponding to 32 mg/m 3 . Thus, damage to the embryo or foetus is unlikely when the MAK value is observed and DEHP is classified in Pregnancy Risk Group C.

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Phthalates Impair Germ Cell Development in the Human Fetal Testis in Vitro without Change in Testosterone Production

Cited by 168 publications

References 59 publications

Novel molecular targets associated with testicular dysgenesis induced by gestational exposure to diethylhexyl phthalate in the rat: a role for estradiol

Novel molecular targets associated with testicular dysgenesis induced by gestational exposure to diethylhexyl phthalate in the rat: a role for estradiol

Establishment of long-term monolayer cultures of somatic cells from human fetal testes and expansion of peritubular myoid cells in the presence of androgen

Di‐(2‐ethylhexyl)phthalat (DEHP) [MAK Value Documentation in German language, 2016]

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