The disrupting effect of chlormequat chloride on growth hormone is associated with pregnancy.

Since chlormequat chloride is widely applied as a plant growth regulator in agriculture and horticulture, its exposure through food consumption is common. We demonstrated previously that chlormequat chloride exposure during pregnancy led to embryos with bigger sizes associated with higher levels of growth hormone (GH) on gestation day 11 (GD11). However, the dose-effect relationship of chlormequat chloride at a lower dose range was not established, and the underlying mechanisms of its promoting effects on embryonic growth and development were not fully elucidated. To address these, pregnant rats were orally exposed to chlormequat chloride at 0, 0.05, 0.5 and 5 mg/kg.bw from GD0 to 11 and the embryonic growth and growth related hormones were evaluated on GD11. We found that the growth and development of the embryos was significantly promoted in a dose dependent manner by chlormequat chloride. Chlormequat chloride also increased embryonic GH, GH releasing hormone (GHRH), and somatostatin (SRIF), and inhibited the embryonic cAMP dependent protein kinase A (PKA) signaling pathway. Chlormequat chloride increased GH synthesis modulated by GHRH/SRIF-PKA-Pituitary specific transcription factor 1 (Pit-1) in the maternal rats. Intriguingly, chlormequat chloride did not show any effects on GH and PKA signaling pathways in the non-pregnant female rats. These findings together suggest that the disrupting effect of chlormequat chloride on GH is associated with pregnancy.

Effects of prenatal chlorocholine chloride exposure on pubertal development and reproduction of male offspring in rats.

Chlorocholine chloride (CCC) promote plant growth as a regulator. Emerging evidence by our group showed that CCC might restrain the puberty onset and impair the reproductive functions in male rats through HPT axis. In this study, we further investigated the effects of prenatal CCC exposure on pubertal development, reproduction of male offspring in rats and explored the underlying mechanisms. The results showed that CCC of 137.5 and 200 mg/kg bw/day delayed the age of preputial separation (PPS), decreased the sperm motility of male offspring. PP1γ2 which is an essential protein in spermatogenesis reduced in 137.5 and 200 mg/kg bw/day groups. Crucial hormones involved in hypothalamic-puititary-testicular (HPT) axis decreased at postnatal day (PND) 30. It was indicated that CCC exposure in pregnancy might disturb the pubertal development, reproductive functions of male offspring through HPT axis and disturb the sperm motility through PP1γ2.

Maternal chlormequat chloride exposure disrupts embryonic growth and produces postnatal adverse effects.

We showed previously that chlormequat chloride, a widely used plant growth regulator, could affect embryonic growth and growth hormone (GH)-insulin-like growth factor 1 (IGF-1) axis of rats. However, the potential effects of low dose chlormequat chloride exposure during pregnancy on embryonic and postnatal growth and development remain unclear. To further assess the risk of chlormequat chloride to human embryonic growth and postnatal health, we exposed maternal rats orally to the chemical during pregnancy at 5 mg/kg bw, a dose corresponding to the human acceptable daily intake (ADI) level set by World Health Organization (WHO), and determined the effects of chlormequat on embryo growth and postnatal health. We found that chlormequat chloride increased embryonic growth parameters, GH, and GH-releasing hormone (GHRH) levels, but did not affect somatostatin and IGF-1 on gestational day (GD) 11. In the pups of postnatal day (PD) 7, we observed increased head length, decreased body fat percentage, hypoglycemia, hyperlipidemia and hyperproteinemia. In conclusion, maternal exposure to chlormequat chloride during pregnancy disrupts the embryonic growth probably through its effects on growth regulators and even has adverse effects on postnatal health.

Effects of chlorocholine chloride on pubertal development and reproductive functions in male rats.

Chlorocholine chloride (CCC), a plant growth retardant, may act as an endocrine disruptor. Our previous study showed that pubertal CCC exposure in rats might decrease testosterone (T) synthesis. This study observed the changes in pubertal development and reproduction of male rats exposed to CCC and its underlying mechanisms. Rats were exposed to CCC (0, 75, 137.5 and 200 mg/kg bw/day) from postnatal day 23 to 60. The results showed that CCC treatment delayed the onset of puberty and reduced the relative organ weight of prostate. Seminiferous tubules with deciduous spermatogenic cells were observed in the 200 mg/kg bw/day group. Sexual behavior was inhibited in the 137.5 and 200 mg/kg bw/day groups. Sperm motility, litter size and normalized anogenital distance (AGD) of male pups were decreased in the 137.5 and 200 mg/kg bw/day groups. Serum kisspeptin level and serum and testicular levels of T were reduced in all CCC treated groups. Crucial hormones in hypothalamic-pituitary-testicular (HPT) axis were reduced subsequently after CCC treatment. Collectively, our results demonstrated that CCC might disturb HPT axis through suppressing the secretion of kisspeptin and subsequently lead to delayed puberty onset and impaired reproductive functions.

Chlormequat chloride promotes rat embryonic growth and GH-IGF-1 axis.

Chlormequat chloride, a plant growth regulator, is widely applied in agriculture because it can promote sturdier growth of the crops. In this research, we found that rat embryo growth on GD11 was inhibited in vitro at 50 µg/ml but promoted in vivo at 75 mg/kg.bw by maternal oral exposure. Therefore, the concentrations of chlormequat chloride in the sera of the pregnant rats on gestation day (GD)11 were determined by a high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) test to be 1.94 ± 0.023 µg/ml, 3.84 ± 0.080 µg/ml, and 7.08 ± 0.11 µg/ml, respectively, when the pregnant rats were orally exposed to chlormequat chloride at 75, 137.5, and 200 mg/kg.bw. Hence, we performed WEC tests again and confirmed that the rat embryo growth in vitro was promoted by chlormequat chloride at 5 µg/mL. The embryonic growth hormone (GH) and insulin-like growth factor 1 (IGF-1) levels were increased by chlormequat chloride both in vitro and in vivo compared with the control ones. We concluded that chlormequat chloride could elevate GH and IGF-1 levels in embryos and promote embryonic growth both in vitro and in vivo.

Chlormequat chloride retards rat embryo growth in vitro.

Chlormequat chloride is the most widely used plant growth regulator in agriculture to promote sturdier growth of grain crops by avoidance of lodging. Therefore, human exposure to chlormequat chloride is very common, but its developmental toxicity has not been studied. Thus, we investigated the developmental toxicity of chlormequat chloride by applying rat whole embryo culture (WEC) model, limb bud micromass culture and 3T3 fibroblast cytotoxicity test. Chlormequat chloride at 150µg/ml (0.93mM) retarded the rat embryo growth without causing significant morphological malformations and at 500µg/ml (3.1mM) caused both retardation and morphological malformation of the embryos. However, the proliferation and differentiation of limb bud cells were not affected by chlormequat chloride at as high as up to 1000µg/ml (6.2mM) applied. This concentration of chlormequat chloride did not affect the cell viability as examined by 3T3 fibroblast cytotoxicity test either, suggesting that cellular toxicity may not play a role in chlormequat induced inhibition of rat embryo growth. Collectively, our results demonstrated that chlormequat chloride may affect embryo growth and development without inhibiting cell viability.