A Novel Concept of Amino Acid Supplementation to Improve the Growth of Young Malnourished Male Rats.

BACKGROUNDS/AIMS: This study was aimed at understanding the relationship between plasma amino acids and protein malnutrition and at determining whether amino acid supplementation associated with malnutrition and growth improves linear growth in growing rats. METHODS: Body length and plasma amino acids were measured in young male rats that were fed the following diet for 3 weeks, mimicking a low and imbalanced protein diets based on maize, a major staple consumed in developing countries: a 70% calorically restricted cornmeal-based diet (C), C + micronutrients (CM), CM + casein (CMC), CM + soy protein (CMS) or CMS + 0.3% lysine. RESULTS: A correlation analysis of linear growth and plasma amino acids indicated that lysine, tryptophan, branched-chain amino acids, methionine, and phenylalanine significantly correlated with body length. Supplementation with these 5 amino acids (AA1) significantly improved the body length in rats compared to CMC treatment whereas, nitrogen-balanced amino acid supplemented controls (AA2) did not (CM +1.2 ± 0.2, CMC +2.7 ± 0.3, CMS +2.1 ± 0.3, AA1 +2.8 ± 0.2, and AA2 +2.5 ± 0.3 cm). CONCLUSION: With securing proper amino acid balance, supplementing growth-related amino acids is more effective in improving linear growth in malnourished growing male rats. Analysis of the correlation between plasma amino acids and growth represents a powerful tool to determine candidate amino acids for supplementation to prevent malnutrition. This technology is adaptable to children in developing countries.

Nutrient Profiling of Japanese Dishes: The Development of a Novel Ajinomoto Group Nutrient Profiling System.

Government agencies and private companies have supported the development of nutrient profiling (NP) systems to facilitate the selection of nutrient-dense foods by consumers, promote nutritious food development, and limit excessive advertising of products with low nutritional value. While most NP models were developed to assess individual foods, the Ajinomoto Group Nutrient Profiling System (ANPS) was developed to assess the overall nutritional value of cooked dishes that are culturally specific to Japan. Based on the national dietary recommendations and nutritional surveys, target values were created for 13 dish categories, while considering the combinations of meal units. For the ANPS, the four evaluating elements were protein and vegetables, which should be encouraged, and sodium and saturated fatty acids, which should be limited. The ANPS algorithm for dishes was the sum of the scores of individual elements, with a maximum of 10 points per serving. The sum of scores was then multiplied by 2.5 to convert to the 100-point scale. Convergent validity was tested using the nutrient-rich food index (NRF) score of 6.3. In total, 1,089 popular Japanese dishes were evaluated using the ANPS, and the median score of ANPS was 70.0 points (interquartile range, 55-78.8), and the average score was 67.7 (standard deviation, 16.5) points. Since salt intake is a major health risk in Japan, this tool was designed to evaluate sodium content with high sensitivity, and low-salt dishes significantly improved sodium and ANPS scores compared with regular dishes. The Pearson's correlation coefficient between the total score of NRF 6.3 and ANPS in 1,089 dishes was r = 0.452 (p < 0.0001). This newly developed ANPS could be used to evaluate culture-specific cooked dishes per serving size. It can determine the nutritional values of dishes, with a high sensitivity to sodium content, a major Japanese nutritional issue. Further research is needed to determine the accuracy and usefulness of the ANPS as a system that would lead to changes in eating behavior nationwide.

Endomucin, a CD34-like sialomucin, marks hematopoietic stem cells throughout development.

To detect as yet unidentified cell-surface molecules specific to hematopoietic stem cells (HSCs), a modified signal sequence trap was successfully applied to mouse bone marrow (BM) CD34(-)c-Kit(+)Sca-1(+)Lin(-) (CD34(-)KSL) HSCs. One of the identified molecules, Endomucin, is an endothelial sialomucin closely related to CD34. High-level expression of Endomucin was confined to the BM KSL HSCs and progenitor cells, and, importantly, long-term repopulating (LTR)-HSCs were exclusively present in the Endomucin(+)CD34(-)KSL population. Notably, in the yolk sac, Endomucin expression separated multipotential hematopoietic cells from committed erythroid progenitors in the cell fraction positive for CD41, an early embryonic hematopoietic marker. Furthermore, developing HSCs in the intraembryonic aorta-gonad-mesonephros (AGM) region were highly enriched in the CD45(-)CD41(+)Endomucin(+) fraction at day 10.5 of gestation (E10.5) and in the CD45(+)CD41(+)Endomucin(+) fraction at E11.5. Detailed analyses of these fractions uncovered drastic changes in their BM repopulating capacities as well as in vitro cytokine responsiveness within this narrow time frame. Our findings establish Endomucin as a novel cell-surface marker for LTR-HSCs throughout development and provide a powerful tool in understanding HSC ontogeny.

Catechin-rich grape seed extract supplementation attenuates diet-induced obesity in C57BL/6J mice.

BACKGROUND: Grape seed extracts (GSE) are known to present health benefits such as antioxidative and anti-obesity effects in animal models. The purpose of this research is to determine whether the specially manufactured GSE, catechin-rich GSE (CGSE), can protect against obesity induced by a high-fat diet (HFD) and to address the mechanism underlying this effect. METHODS: The componential analysis of CGSE was performed using liquid chromatography/mass spectrometry. Oxygen consumption and the respiratory quotient were determined using 500 mg/kg CGSE administered orally for 3 days in 14- to 15-week-old male C57BL/6J mice. Nine-week-old male C57BL/6J mice were supplemented with 0.5 or 1% CGSE in a HFD for 12 weeks, and their body weight and food intake were monitored. Blood and tissue samples were collected and analyzed. RESULTS: The main polyphenol components of CGSE were catechin and epicatechin. CGSE supplementation in the HFD-induced obesity model chronically suppressed the increase in body weight and the weight of fat pads. Furthermore, CGSE improved metabolic parameter abnormalities and upregulated the fatty acid oxidation-related genes in the liver. CONCLUSIONS: These findings suggest that CGSE contains monomeric catechins in high concentrations and ameliorates HFD-induced obesity in C57BL/6J mice.

Changes of Plasma Amino Acid Profiles in Infants With a Nutrient-Fortified Complementary Food Supplement: Evidence From a 12-Month Single-Blind Cluster-Randomized Controlled Trial.

Stunting is reportedly associated with low circulating levels of essential amino acids (EAAs). This study examined the effect of a macronutrient- and micronutrient-fortified complementary food supplement (KOKO Plus) on specific plasma EAA levels and stunting in infants aged 6-18 months. In a single-blind cluster-randomized controlled trial conducted in Ghana, infants were enrolled at 6 months and followed until 18 months. Thirty-eight communities were randomly assigned to receive KOKO Plus (KP, fourteen communities, n = 321), multiple-micronutrient powder (MN, thirteen communities, n = 327), or only nutritional education as control group (NE, eleven communities, n = 318), and all groups received nutrition education. Plasma amino acids (AAs) were measured at 6, 12, and 18 months (end point). Mixed-effects models were used to assess the effect of the intervention on plasma AAs, and the relationship between plasma branched-chain AAs (BCAAs) and the risk of stunting was assessed. At the end point, total BCAA concentrations (±standard error) significantly exceeded baseline in the KP (284.2 ± 4.3 µM) and NE (289.1 ± 4.4 µM) groups but not the MN group (264.4 ± 4.1 µM). After adjustment for compliance at 200 sachets, plasma BCAAs exceeded in the KP group (284.5 ± 4.2 µM) compared to the MN group (264.6 ± 4 µM). Plasma BCAAs were positively correlated with changes in length-for-age Z-score from baseline (R = 0.327, p = 0.048). In conclusion, the plasma BCAA concentrations of infants that received KP and the NE group was significantly higher compared to the MN group but there were no differences between the KP and NE group at end point. Improved plasma BCAAs may be due to improved nutrient intake by infants exposed to KP or NE. Low BCAAs were associated with stunting, replicating the previous finding. Clinical Trial Registration: https://clinicaltrials.gov/ct2/show/NCT03181178?term=NCT03181178&draw=2&rank=1, identifier: NCT03181178.

Amino acid-enriched plant-based RUTF treatment was not inferior to peanut-milk RUTF treatment in restoring plasma amino acid levels among patients with oedematous or non-oedematous malnutrition.

Ready-to-use therapeutic food (RUTF) with adequate quality protein is used to treat children with oedematous and non-oedematous severe acute malnutrition (SAM). The plasma amino acid (AA) profile reflects the protein nutritional status; hence, its assessment during SAM treatment is useful in evaluating AA delivery from RUTFs. The objective was to evaluate the plasma AAs during the treatment of oedematous and non-oedematous SAM in community-based management of acute malnutrition (CMAM) using amino acid-enriched plant-based RUTFs with 10% milk (MSMS-RUTF) or without milk (FSMS-RUTF) compared to peanut milk RUTF (PM-RUTF). Plasma AA was measured in a non-blinded, 3-arm, parallel-group, simple randomized controlled trial conducted in Malawi. The RUTFs used for SAM were FSMS-RUTF, MSMS-RUTF or PM-RUTF. A non-inferiority hypothesis was tested to compare plasma AA levels from patients treated with FSMS-RUTF or MSMS-RUTF with those from patients treated with PM-RUTF at discharge. For both types of SAM, FSMS-RUTF and MSMS-RUTF treatments were non-inferior to the PM-RUTF treatment in restoration of the EAA and cystine except that for FSMS-RUTF, methionine and tryptophan partially satisfied the non-inferiority criteria in the oedematous group. Amino-acid-enriched milk-free plant-source-protein RUTF has the potential to restore all the EAA, but it is possible that enrichment with amino acids may require more methionine and tryptophan for oedematous children.

Antioxidative activities of Oxindole-3-acetic acid derivatives from supersweet corn powder.

The components contributing to the antioxidative activity of supersweet corn powder (SSCP), which is commonly used in corn soup and snacks in Japan, were clarified and the effects investigated. 7-(O-ß-Glucosyloxy)oxindole-3-acetic acid (GOA) was found to be the component most strongly contributing to the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity of the 80% ethanol extract of SSCP, and the presence of its aglycone, 7-hydroxy-oxindole-3-acetic acid (HOA) was confirmed. GOA and HOA respectively contributed 35.1% and 10.5% to the DPPH radical-scavenging activity of the 80% ethanol extract of SSCP. Mice orally administered with HOA at doses of both 500 and 1500 mg/kg showed a significantly lower (p<0.05) level of thiobarbituric acid reactive substances (TBARS) in the plasma than the vehicle-treated control. These results suggest that GOA and HOA were at least partly involved in the antioxidative activity of SSCP in vitro and that HOA might have possessed antioxidative activity in vivo.

A macro- and micronutrient-fortified complementary food supplement reduced acute infection, improved haemoglobin and showed a dose-response effect in improving linear growth: a 12-month cluster randomised trial.

Inadequate protein quality may be a risk factor for poor growth. To examine the effect of a macronutrient-micronutrient supplement KOKO Plus (KP), provided to infants from 6 to 18 months of age, on linear growth, a single-blind cluster-randomised study was implemented in Ghana. A total of thirty-eight communities were randomly allocated to receive KP (fourteen communities, n 322), a micronutrient powder (MN, thirteen communities, n 329) and nutrition education (NE, eleven communities, n 319). A comparison group was followed cross-sectionally (n 303). Supplement delivery and morbidity were measured weekly and anthropometry monthly. NE education was provided monthly. Baseline, midline and endline measurements at 6, 12 and 18 months included venous blood draws, diet, anthropometry, morbidity, food security and socio-economics. Length-for-age Z-score (LAZ) was the primary outcome. Analyses were intent-to-treat using mixed-effects regressions adjusted for clustering, sex, age and baseline. No differences existed in mean LAZ scores at endline (-1·219 (sd 0·06) KP, -1·211 (sd 0·03) MN, -1·266 (sd 0·03) NE). Acute infection prevalence was lower in the KP than NE group (P = 0·043). Mean serum Hb was higher in KP infants free from acute infection (114·02 (sd 1·87) g/l) than MN (107·8 (sd 2·5) g/l; P = 0·047) and NE (108·8 (sd 0·99) g/l; P = 0·051). Compliance was 84·9 % (KP) and 87·2 % (MN) but delivery 60 %. Adjusting for delivery and compliance, LAZ score at endline was significantly higher in the KP v. MN group (+0·2 LAZ; P = 0·026). A macro- and micronutrient-fortified supplement KP reduced acute infection, improved Hb and demonstrated a dose-response effect on LAZ adjusting consumption for delivery.

Amino-acid-enriched cereals ready-to-use therapeutic foods (RUTF) are as effective as milk-based RUTF in recovering essential amino acid during the treatment of severe acute malnutrition in children: An individually randomized control trial in Malawi.

BACKGROUND: Ready-to-use therapeutic food (RUTF) is used to treat children suffering from severe acute malnutrition (SAM). Standard RUTF uses milk as the primary protein source, which makes the product expensive, and given the high worldwide SAM burden, having a less expensive effective alternative is a public health priority. OBJECTIVE: The objective of this study was to evaluate whether newly developed amino acid-enriched milk-free RUTF (FSMS-RUTF) or amino acid-enriched low-milk RUTF (MSMS-RUTF) treatment could replenish plasma amino acids to levels comparable to those following standard peanut-milk RUTF (PM-RUTF) treatment and to improve understanding of the effects of treatment on anthropometric measurements. A secondary analysis was performed to test the noninferiority hypothesis of plasma essential amino acid (EAA) levels. METHODS: Plasma EAA levels were measured in a nonblinded, 3-arm, parallel-group simple randomized controlled trial conducted in Malawi to examine the efficacy of FSMS-RUTF, MSMS-RUTF and PM-RUTF in the treatment of SAM in 2 groups of children aged 6-23 and 24-59 months (mo). Sample size calculations were performed based on the previous our study. A noninferiority margin was set at -25% of the PM-RUTF arm at discharge. RESULTS: The relative values of the differences (95% CI) in plasma EAA levels between PM-RUTF treatment and FSMS-RUTF and MSMS-RUTF treatments at discharge were -7.9% (-18.6, 2.8) and 9.8% (0.2, 19.5), respectively, in children aged 6-23 mo, while in those aged 24-59 mo, the difference values were 17.8% (1.6, 34.1) and 13.6% (-2.8, 29.9), respectively. CONCLUSION: At discharge, the plasma EAA concentrations in 6-59-mo-old SAM children treated with FSMS-RUTF and MSMS-RUTF were not less than those of children treated with PM-RUTF. These findings indicate that treatment with either of the 3 RUTFs was associated with adequate protein synthesis and that all the formulations provided sufficient functional metabolites of plasma amino acids to support nutritional recovery from SAM.

Effects of 3-methyl-4-nitrophenol in diesel exhaust particles on the regulation of testicular function in immature male rats.

We investigated the effects of 3-methyl-4-nitrophenol (4-nitro-m-cresol, PNMC) isolated from diesel exhaust particles (DEP) on the reproductive functions of male rats. Twenty-eight-day-old rats were injected subcutaneously with PNMC (1, 10, or 100 mg/kg) daily for 5 days. The weights of the epididymis, seminal vesicle, and Cowper gland were significantly decreased in rats treated with 10 mg/kg PNMC. The plasma concentrations of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were significantly increased by PNMC at 100 mg/kg. However, the plasma concentrations of testosterone and immunoreactive (ir)-inhibin were significantly decreased by PNMC at 100 mg/kg. The testosterone content of the testicles was significantly decreased in the group treated with 100 mg/kg PNMC compared with the control group. Furthermore, testicular concentration of ir-inhibin was significantly decreased by PNMC at 1 mg/kg or 100 mg/kg. To investigate the direct effects of PNMC on the secretion of LH and FSH from the anterior pituitary gland, and on the secretion of testosterone from the testes, we exposed cultured anterior pituitary and interstitial Leydig cells to PNMC (10(-6), 10(-5), 10(-4) M) with or without gonadotropin-releasing hormone (GnRH; 10 nM) (for the LH and FSH tests) and human chorionic gonadotropin (hCG; 0.1 IU/mL) (for the testosterone test) for 24 hours. PNMC did not change either the basal or GnRH-stimulated levels of FSH and LH secretion. However, PNMC significantly inhibited both basal and hCG-stimulated testosterone production. These findings suggest that PNMC has a direct effect on the testes of immature male rats, causing a reduction in testosterone secretion.

Impairment of testicular function in adult male Japanese quail (Coturnix japonica) after a single administration of 3-methyl-4-nitrophenol in diesel exhaust particles.

The effects of 3-methyl-4-nitrophenol (PNMC), a component of diesel exhaust, on reproductive function were investigated in adult male Japanese quail. The quail were treated with a single i.m. dose of PNMC (78, 103 or 135 mg/kg body weight), and trunk blood and testes were collected 1, 2 or 4 weeks later. Various levels of testicular atrophy were observed in all groups treated with PNMC. Sperm formation, cloacal gland area, and plasma LH and testosterone concentrations were also reduced in birds with testicular atrophy. To determine the acute effect of PNMC on gonadotrophin from the pituitary, adult male quail were administrated a single i.m. injection of PNMC (25 mg/kg), and plasma concentrations of LH were measured at 1, 3 and 6 h. This dose significantly lowered plasma levels of LH at all three time points. These results suggest that PNMC acts on the hypothalamus-pituitary axis, by reducing circulating LH within a few hours of administration and subsequently reducing testosterone secretion. In addition, in order to investigate the direct effects of PNMC on the secretion of testosterone from testicular cells in quail testes, cultured interstitial cells containing Leydig cells were exposed to PNMC (10(-6), 10(-5) or 10(-4) M) for 4, 8 or 24 h. These quantities of PNMC significantly reduced the secretion of testosterone in a time- and dose-dependent manner. The present findings also suggest a direct effect of PNMC on the testis to reduce testosterone secretion. This study clearly indicates that PNMC induces reproductive toxicity at both the central and testicular levels, and disrupts testicular function in adult male quail.

Anti-androgenic activity of 3-methyl-4-nitrophenol in diesel exhaust particles.

In our continuing studies on nitrophenol derivatives as vasodilators in diesel exhaust particles, we have reported that nitrophenols in diesel exhaust particles possess not only vasodilatory activity but also estrogenic activity in vitro and in vivo, as well as anti-androgenic activity in vitro. Our efforts here were focused on the in vitro and in vivo anti-androgenic activity of 3-methyl-4-nitrophenol (4-nitro-m-cresol; PNMC), known a degradation product of the insecticide fenitrothion, in diesel exhaust particles. We investigated its anti-androgenic activity using an in vitro recombinant yeast screen and in vivo Hershberger assays. Recombinant yeast screen assay showed that PNMC possesses anti-androgenic activity at low concentrations. Furthermore, castrated 28-day-old immature male rats each implanted with a 5-mm-long silastic tube containing crystalline testosterone and injected with PNMC subcutaneously at doses from as low as 0.01 and 0.1 mg/kg up to 1 mg/kg for 5 consecutive days showed significantly decreased weights of the seminal vesicles, ventral prostate, and glans penis. Plasma follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels were significantly increased in the 0.1 mg/kg PNMC treatment group. Our results demonstrate that PNMC in diesel exhaust particles clearly has anti-androgenic activity both in vitro and in vivo and can therefore be considered as an endocrine-disrupting chemical.

Expression of nerve growth factor (NGF), and its receptors TrkA and p75 in the reproductive organs of the adult male rats.

Immunolocalization of nerve growth factor (NGF) and its receptors, TrkA and p75 in the reproductive organs of adult male rats was investigated. Sections of the testis, efferent duct, epididymis, deferent duct, seminal vesicle, coagulating gland and prostate of adult male rats were immunostained by the avidin-biotin-peroxidase complex methods (ABC). NGF was expressed in Leydig cells, primary spermatocytes and pachytene spermatocytes in the testis. TrkA only immunoreacted to elongate spermatids and p75 showed positive immunostaining in the Sertoli cells, Leydig cells, the pachytene spermatocytes and elongate spermatids. Immunoreactions for NGF and its two receptors were detected in epithelial cells of efferent duct, deferent duct and epididymis. In addition, immunoreactions for NGF and its two receptors were also observed in columnar secretory epithelium lines of the seminal vesicles, prostate and coagulating gland. These results suggest that NGF is an important growth factor in gonadal function of adult male rats.

Combined supplementation of carbohydrate, alanine, and proline is effective in maintaining blood glucose and increasing endurance performance during long-term exercise in mice.

Carbohydrate supplementation is extremely important during prolonged exercise because it maintains blood glucose levels during later stages of exercise. In this study, we examined whether maintaining blood glucose levels by carbohydrate supplementation could be enhanced during long-term exercise by combining this supplementation with alanine and proline, which are gluconeogenic amino acids, and whether such a combination would affect exercise endurance performance. Male C57BL/6J mice were orally administered either maltodextrin (1.25 g/kg) or maltodextrin (1.0 g/kg) with alanine (0.225 g/kg) and proline (0.025 g/kg) 15 min before running for 170 min. Combined supplementation of maltodextrin, alanine, and proline induced higher blood glucose levels than isocaloric maltodextrin alone during the late exercise phase (100-170 min). The hepatic glycogen content of mice administered maltodextrin, alanine, and proline was higher than that of mice ingesting maltodextrin alone 60 min after beginning exercise, but the glycogen content of the gastrocnemius muscle showed no difference. We conducted a treadmill running test to determine the effect of alanine and proline on endurance performance. The test showed that running time to exhaustion of mice that were supplemented with maltodextrin (2.0 g/kg) was longer than that of mice that were supplemented with water alone. Maltodextrin supplementation (1.0 g/kg) with alanine (0.9 g/kg) and proline (0.1 g/kg) further increased running time to exhaustion compared to maltodextrin alone (2.0 g/kg). These results indicate that combined supplementation of carbohydrate, alanine, and proline is effective for maintaining blood glucose and hepatic glycogen levels and increasing endurance performance during long-term exercise in mice.

Improving complementary feeding in Ghana: reaching the vulnerable through innovative business--the case of KOKO Plus.

Reaching vulnerable populations in low-resource settings with effective business solutions is critical, given the global nature of food and nutrition security. Over a third of deaths of children under 5 years of age are directly or indirectly caused by undernutrition. The Lancet series on malnutrition (2013) estimates that over 220,000 lives of children under 5 years of age can be saved through the implementation of an infant and young child feeding and care package. A unique project being undertaken in Ghana aims to bring in two elements of innovation in infant and young child feeding. The first involves a public-private partnership (PPP) to develop and test the efficacy and effectiveness of the delivery of a low-cost complementary food supplement in Ghana called KOKO Plus™. The second involves the testing of the concepts of social entrepreneurship and social business models in the distribution and delivery of the product. This paper shares information on the ongoing activities in the testing of concepts of PPPs, social business, social marketing, and demand creation using different delivery platforms to achieve optimal nutrition in Ghanaian infants and young children in the first 2 years of life. It also focuses on outlining the concept of using PPP and base-of-the-pyramid approaches toward achieving nutrition objectives.

Placental activin A is required for follicular development during the second half of pregnancy in the golden hamster (Mesocricetus auratus).

Numerous antral follicles develop during the second half of pregnancy in the golden hamster even though LH and FSH are maintained at basal levels. To investigate the possible hormone actions of activin A associated with follicular development, pregnant golden hamsters were placentectomized on day 6 of pregnancy and animals were sacrificed at day 8, 10, 12, or 14 of pregnancy. There was a drastic decrease in the plasma concentrations of activin A from day 10 of pregnancy in the operated group compared to the controls. Positive immunohistochemical staining of inhibin/activin subunits betaA and betaB in the syncytiotrophoblast of the placenta revealed the source of activin A, AB, or B. The number of healthy follicles did not change until day 12 between the operated and the control groups, but decreased in numbers in the operated groups thereafter. The decreased concentrations of inhibin A, B, and estradiol-17beta in the operated groups at day 10 and 12 correlated well with the number of mature follicles in response to hCG treatment. In conclusion, we revealed that activin A secreted from the placenta induces folliculogenesis to maintain the high levels of estradiol-17beta needed to induce uterine dilatation for fetus growth and impending parturition.

Comparative effects of prolactin versus ACTH, estradiol, progesterone, testosterone, and dihydrotestosterone on cortisol release and proliferation of the adrenocortical carcinoma cell line H295R.

In this study, using the H295R cell line as a model system, we investigated the role of prolactin (PRL) and steroid hormones in the growth regulation and cortisol release of adrenocortical cells. H295R cells were treated with increasing doses (10(-13)-10(-6) M) of PRL, adrenocorticotropic hormone (ACTH), 17beta-estradiol (E(2)), progesterone (P(4)), testosterone (T), and dihydrotestosterone (DHT). As expected, ACTH raised cortisol secretion and increased the proliferation rate of cultured cells. Incubation with T, DHT, E(2), and P(4) for 24 h did not significantly increase cortisol release. Conversely, PRL concentrations of 10(-8)-10(-6) M caused a significant increase in the release of cortisol. Long-term (5 days) stimulation of H295R cells with E(2), P(4), and PRL was a trigger to increased cell proliferation, while T and DHT did not alter H295R cell proliferation. Taken together, these results indicate that steroid hormones exert differential effects on adrenocortical function. Additionally, the present study demonstrates that PRL had biphasic actions in regulating adrenocortical function. PRL may form a novel regulatory system for steroid hormone secretion and cell proliferation in the adrenal cortex.

Nitrophenols isolated from diesel exhaust particles regulate steroidogenic gene expression and steroid synthesis in the human H295R adrenocortical cell line.

Studies of nitrophenols isolated from diesel exhaust particles (DEPs), 3-methyl-4-nitrophenol (PNMC) and 4-nitro-3-phenylphenol (PNMPP) have revealed that these chemicals possess estrogenic and anti-androgenic activity in vitro and in vivo and that PNMC accumulate in adrenal glands in vivo. However, the impacts of exposure to these compounds on adrenal endocrine disruption and steroidogenesis have not been investigated. To elucidate the non-receptor mediated effects of PNMC and PNMPP, we investigated the production of the steroid hormones progesterone, cortisol, testosterone, and estradiol-17beta and modulation of nine major enzyme genes involved in the synthesis of steroid hormones (CYP11A, CYP11B1, CYP17, CYP19, 17betaHSD1, 17betaHSD4, CYP21, 3betaHSD2, StAR) in human adrenal H295R cells supplied with cAMP. Exposure to 10(-7) to 10(-5) M PNMC and 1 mM 8-Br-cAMP for 48 h decreased testosterone, cortisol, and estradiol-17beta levels and increased progesterone secretion. At 10(-5) M, PNMC with 1 mM 8-Br-cAMP significantly stimulated expression of the 17betaHSD4 and significantly suppressed expression of 3betaHSD2. In comparison, 10(-7) to 2 x 10(-5) M PNMPP with 1 mM 8-Br-cAMP for 48 h decreased concentrations of estradiol-17beta, increased progesterone levels, but did not affect testosterone and cortisol secretion due to the significant suppression of CYP17 and the non-significant but obvious suppression of CYP19. Our results clarified steroidogenic enzymes as candidates responsible for the inhibition or stimulation for the production of steroid hormones in the steroidogenic pathway, thus providing the first experimental evidence for multiple mechanisms of disruption of endocrine pathways by these nitrophenols.

Nitrophenols isolated from diesel exhaust particles promote the growth of MCF-7 breast adenocarcinoma cells.

Diesel exhaust particles (DEPs) cause many adverse health problems, and reports indicate increased risk of breast cancer in men and women through exposure to gasoline and vehicle exhaust. However, DEPs include vast numbers of compounds, and the specific compound(s) responsible for these actions are not clear. We recently isolated two nitrophenols from DEPs-3-methyl-4-nitrophenol (4-nitro-m-cresol; PNMC) and 4-nitro-3-phenylphenol (PNMPP)-and showed that they had estrogenic and anti-androgenic activities. Here, we tried to clarify the involvement of these two nitrophenols in promoting the growth of the MCF-7 breast cancer cell line. First, comet assay was used to detect the genotoxicity of PNMC and PNMPP in a CHO cell line. At all doses tested, PNMC and PNMPP showed negative genotoxicity, indicating that they had no tumor initiating activity. Next, the estrogen-responsive breast cancer cell line MCF-7 was used to assess cell proliferation. Proliferation of MCF-7 cells was stimulated by PNMC, PNMPP, and estradiol-17beta and the anti-estrogens 4-hydroxytamoxifen and ICI 182,780 inhibited the proliferation. To further investigate transcriptional activity through the estrogen receptor, MCF-7 cells were transfected with a receptor gene that allowed expression of luciferase enzyme under the control of the estrogen regulatory element. PNMC and PNMPP induced luciferase activity in a dose-dependent manner at submicromolar concentrations. ICI 182,780 inhibited the luciferase activity induced by PNMC and PNMPP. These results clearly indicate that PNMC and PNMPP do not show genotoxicity but act as tumor promoters in an estrogen receptor alpha-predominant breast cancer cell line.

Effects of 3-methyl-4-nitrophenol in diesel exhaust particles on the regulation of reproductive function in immature female Japanese quail (Coturnix japonica).

In a previous study, we found that 3-methyl-4-nitrophenol (PNMC), a component of diesel exhaust particles and also a degradation product of the insecticide fenitrothion, exhibits reproductive toxicity in the adult male Japanese quail. The present study investigated the toxicity of PNMC in the female Japanese quail and its ability to influence reproduction in immature females. The quail (21-day-old) were injected intramuscularly (im) with PNMC at doses 0.1, 1 or 10 mg/kg body weight daily for 3 days. There was no significant difference in body growth between the PNMC-administered and control birds. However, the weights of the oviducts were significantly lower in the PNMC-treated birds at all doses. Furthermore, the plasma concentrations of luteinizing hormone (LH) and estradiol-17 beta were significantly decreased with 1 and 10 mg/kg of PNMC. These findings suggest that PNMC might influence the hypothalamo-pituitary-gonadal axis with decreasing in secretion of GnRH, LH and ovarian steroid hormones and subsequently disturb growth of the reproductive organs of immature female quail. This study indicates that PNMC induces reproductive toxicity at the central level and disrupts reproductive function in the immature female quail.