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. Author manuscript; available in PMC: 2016 Jan 31.
Published in final edited form as: Birth Defects Res B Dev Reprod Toxicol. 2015 Feb;104(1):35–51. doi: 10.1002/bdrb.21137

Effects of maternal and lactational exposure to 2-hydroxy-4-methoxybenzone on development and reproductive organs in male and female rat offspring

Noriko Nakamura 1, Amy L Inselman 1, Gene A White 1, Ching-Wei Chang 2, Raul A Trbojevich 3, Estatira Sepehr 3, Kristie L Voris 4, Ralph E Patton 4, Matthew S Bryant 3, Wafa Harrouk 5, Barry McIntyre 6, Paul M Foster 6, Deborah K Hansen 1
PMCID: PMC4353586  NIHMSID: NIHMS661100  PMID: 25707689

Abstract

BACKGROUND

2-hydroxy-4-methoxybenzophenone (HMB) is an ultraviolet (UV)-absorbing compound used in many cosmetic products as a UV-protecting agent and in plastics for preventing UV-induced photodecomposition. HMB has been detected in over 95% of randomly collected human urine samples from adults and from premature infants, and it may have estrogenic potential.

METHODS

To determine the effects of maternal and lactational exposure to HMB on development and reproductive organs of offspring, time-mated female Harlan Sprague-Dawley rats were dosed with 0, 1,000, 3,000, 10,000, 25,000, or 50,000 ppm HMB (7-8 per group) added to chow from gestation day 6 until weaning on postnatal day (PND) 23.

RESULTS AND CONCLUSION

Exposure to HMB was associated with reduced body and organ weights in female and male offspring. No significant differences were observed in the number of implantation sites/litter, mean resorptions/litter, % litters with resorptions, number and weights of live fetuses, or sex ratios between the control and HMB dose groups. Normalized anogenital distance in male pups at PND 23 was decreased in the highest dose group. Spermatocyte development was impaired in testes of male offspring in the highest dose group. In females, follicular development was delayed in the highest dose group. However, by evaluating levels of the compound in rat serum, the doses at which adverse events occurred are much higher than usual human exposure levels. Thus, exposure to less than 10,000 ppm HMB does not appear to be associated with adverse effects on the reproductive system in rats.

Keywords: 2-hydroxy-4-methoxybenzophenone, body weight, rats, offspring, testes, ovary, fetal exposure

Introduction

2-hydroxy-4-methoxybenzophenone (HMB) (CAS #131-57-7; oxybenzone, or benzophenone-3) is a benzophenone derivative that can absorb ultraviolet (UV) light. HMB and its derivatives are utilized as stabilizers in plastics for preventing UV-induced photodecomposition (Kamogawa, 1969; Dormán and Prestwich, 1994) and used as an active ingredient in sunscreens to protect the skin from both UVA and UVB radiation (Klein, 1992).

The most prominent route of exposure for HMB in humans is through the skin (Janjua et al., 2004; Hayden et al., 2005). HMB has been detected in over 95% of urine samples from human adults collected through NHANES indicating extensive exposure to this compound (Calafat et al., 2008). It has also been detected in urine from premature infants and in human breast milk, indicating that HMB can be transferred to progeny through the placenta or via lactation (Calafat et al., 2009; Schlumpf et al., 2010). The significance of the presence of HMB in human urine is currently unknown since whole-body topical application of HMB does not appear to cause adverse effects in humans (Janjua et al., 2004). Previous animal studies have shown that postnatal exposure to HMB in mice and rats, via dietary or dermal administration alters body, liver and kidney weights in males and females (National Toxicology Program, 1992). In addition, sperm numbers in the cauda epididymis of rats and mice were found to be significantly reduced when exposed to 50,000 ppm HMB in chow for 13 weeks when compared to controls (National Toxicology Program, 1992). Sperm number in mice exposed by dermal administration for 13 weeks was also observed to be significantly decreased in a dose-dependent manner in all dose groups when compared to the controls (National Toxicology Program, 1992). In females, estrous cycle length was significantly longer when 50,000 ppm HMB was added to chow for 13 weeks when compared to controls (National Toxicology Program, 1992). Another study reported that oral postnatal exposure of rats to HMB for 27 days was tolerated, but extension to 90 days reduced body and organ weights and caused kidney abnormalities in male rats, even at doses of 0.5% and 1.0% HMB (Lewerenz et al., 1972; Christian, 1983). However, Daston et al. (1993) found that dermal exposure of mice to HMB had no effect on reproductive parameters; these authors suggested that the positive results observed in the NTP study may have been due, in part, to sperm counts which were higher than their historical control data (Morrissey et al., 1988).

Several in vitro and in vivo studies have suggested that HMB may have estrogenic potential (Nakagawa and Suzuki, 2002; Suzuki et al., 2005). Chemicals with estrogenic activity are known to disrupt normal homeostasis, development, and reproduction (Diamanti-Kandarakis et al., 2009). In male reproduction, these chemicals disrupt spermatogenesis and testicular steroidogenesis (Kuwada et al., 2002; Alam et al., 2010). Recent studies reported that other UV-filters [benzophenone-2 (BP-2) and benzophenone-4] have adverse effects on reproduction and development in fish and mice due to their estrogen-like activity (Kunz et al., 2006; Coronado et al., 2008; Kim et al., 2011; Zucchi et al., 2011). In addition, BP-2 exposure has been reported to inhibit oocyte development in fish (Weisbrod et al., 2007). A recent epidemiological study has indicated that increased levels of an HMB metabolite may be associated with an increased risk of a diagnosis of endometriosis (Kunisue et al., 2012). The authors suggested mechanistic studies needed to be done to clarify the potential endocrine disrupting activity of HMB and its metabolites.

The aim of this study was to obtain preliminary data on the maternally-mediated effects of HMB on development and reproductive organs of rat offspring at PND 23. To examine this, groups of time-mated 11-13-week-old female Harlan Sprague-Dawley rats were administrated dietary doses of HMB from gestation day (GD) 6 until euthanasia at postnatal day (PND) 23. These animals were part of a dose range-finding study that was being conducted prior to guideline studies of oxybenzone; therefore, the number of litters in each treatment group was small.

Materials and Methods

Materials

All reagents were purchased from Fisher Scientific (Pittsburgh, PA, USA), unless otherwise indicated.

Animals and treatments

Groups of 11–13-week-old time-mated female Sprague-Dawley rats were purchased from Harlan Laboratories (Indianapolis, IN, USA) and were delivered to the National Center for Toxicological Research (NCTR) on GD 3 (day of vaginal plug detection designated GD 0). Pregnant dams were housed individually and maintained under a 12:12-h light-dark cycle with controlled room temperature (23°C ± 3°C) and humidity (50% ± 20%). Starting on GD 6 until PND 23, dams were fed low-phytoestrogen chow (Purina 5K96; Purina Mills LLC, St Louis, MO, USA) containing 0, 1,000, 3,000, 10,000, 25,000, or 50,000 ppm HMB (Catalog #HH13-026; Ivy Fine Chemicals, Cherry Hill, NJ, USA); these doses were similar to those (0, 3,125, 6,250, 12,500, 25,000, or 50,000 ppm) used by the National Toxicology Program in a previous adult only exposure study (National Toxicology Program, 1992). Water was provided ad libitum. Food consumption (g/animal/day) was measured at the start of dosing on GD 6 and at GD 10, 13, 20, PND 7, 14 and 23. Animal weights were recorded on GD 3 (delivery date), 6, 10, 13 and 20. The dams were euthanized on GD 10, 15, 20 or PND 23 (n = 7-8 per exposure level per time point). For the animals sacrificed on PND 23, dams were allowed to deliver naturally (day of birth = PND 0) and nurse their pups. On PND 1, litters were culled to 10 pups, five males and five females, whenever possible. Pup body weights were recorded on PND 1, 4, 7, 13 and 23. All animal procedures were approved by the NCTR Institutional Animal Care and Use Committee and followed the Guidelines set forth by the National Research Council’s Guide for the Care and Use of Laboratory Animals (National Research Council, 1996).

Dams

Sample collection

All dams were weighed prior to euthanizing them by over-exposure to carbon dioxide. Blood was collected from cardiac puncture for measuring HMB and its metabolites, and uteri were collected from dams between 8-11 AM on GD 10, 15, and 20. The number of implantation sites and resorptions were recorded from GD 10 and 15 dams. At GD 20, uterine weights were recorded and the number of implantation sites, resorptions, and dead and live fetuses were determined. Fetuses were observed for external malformations, and then dissected to determine their sex. Testes were collected from male fetuses for gene expression analysis. For the PND 23 group, dams were euthanized with carbon dioxide, followed by collection of blood and tissues (liver, paired kidneys, paired ovaries and uteri) between 8-11 AM. Body and organs weights were recorded. Blood was collected by cardiac puncture and transferred into blood collection tubes (BD, Franklin Lakes, NJ, USA). Serum was obtained by centrifugation at 3,000 × g for 10 min at room temperature.

Measurement of HMB and metabolites in serum of dams

Serum levels of HMB and three metabolites [2,4-dihydroxybenzophenone (DHB), 2,3,4-trihydroxybenzophenone (THB) and 2,2-dihydroxy-4-methoxybenzophenone (DHMB)] were measured using HPLC tandem mass spectrometry. Serum (40 μl) was subjected to protein precipitation with 100 μl acetonitrile with 1 μg/ml reserpine as an internal standard (Sigma-Aldrich, St. Louis, MO, USA) (Bryant et al., 1997). After vortexing for 1-2 minutes and centrifuging at 12,000 × g for 8 min, 10 μl supernatant was used for analysis. All sample extracts were maintained in the autosampler at 15 °C while awaiting injection. Chromatographic separation was achieved with a Shimadzu Prominence UFLC (Shimadzu Scientific Instruments, Columbia, MD, USA) using a Synergi 4 μm Fusion- RP 80Å, C18-LC column (2 × 50 mm) and guard column (Phenomenex, Torrance, CA, USA). Initial gradient conditions were set at 5% acetonitrile (ACN) containing 0.1% formic acid and held for 1 min before incorporating a linear gradient increasing to 63% ACN over 6 min and held for 3 min. The flow rate for all analyses was 0.3 ml/min, and the column temperature was 40°C. The analyte was detected using a 3200 QTRAP mass spectrometer (AB SCIEX, Framingham, MA, USA) operated in the electrospray mode. The instrument settings were tuned for maximum sensitivity of the HMB and metabolites using infusion of calibration standards. A multiple reaction monitoring scheme was established to detect each of the four analytes and the internal standard. Serum calibration curves consisting of six concentrations of each analyte were prepared daily and used to calculate the levels in the study samples. The limit of detection (LOD) for HMB, DHB, and DHMB was 0.005 μg/ml, while the LOD for THB was 0.05 to 0.1 μg/ml.

Clinical chemistry analysis

Clinical chemistry analyses including glucose, alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate transaminase (AST), total protein, albumin, cholesterol, triglycerides, inorganic (I) phosphorus, blood urea nitrogen (BUN), and creatinine were performed on an ACE Alera Chemistry system (ALFA Wassermann Diagnostic Technologies, LLC West Caldwell, NJ, USA). Sorbitol dehydrogenase (SDH) and total bile acids (TBA) were measured using VetSpec SDH and Bile Acid Kits, respectively (Catachem, Inc., Oxford, CT, USA). The serum from one female and male pup from each litter was analyzed.

Male and female offspring

Anogenital distance (AGD)

AGD at PND 1 and 23 was measured using a microscope with a reticle eye piece and a correction factor used to translate ocular units to mm. Normalization of AGD to body weight was performed as described by Gallavan et al. (1999).

Sample collection

Male and female offspring at PND 23 were euthanized with carbon dioxide, followed by collection of blood by cardiac puncture and dissection of selected tissues. Body and organ weights (paired testes, paired epididymides, paired ovaries, uteri, liver and paired kidneys) were recorded.

Ovarian histology

Ovaries from all PND 23 female pups were fixed in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) in PBS at 4 °C. Samples were then dehydrated and embedded in paraffin according to standard procedures and cut into 5-μm serial sections. After deparaffinization, slides were stained with hematoxylin QS (Vector Laboratories, Burlingame, CA) for 30 sec, washed in running water, and stained with a 1% eosin/alcohol solution (Sigma-Aldrich) for 2 min. The stained slides were dehydrated and mounted with Poly-Mount (Polyscience Inc., Warrington, PA, USA). Images were captured with a Zeiss AxioVision microscope and software (Carl Zeiss Microscopy, LLC, Thornwood, NY, USA) using the same exposure time for all images. Every fifth section was counted from the first section containing follicular cells until the last sections without follicular cells were observed. Only follicular cells that contained an oocyte with a nucleus were counted. Follicular cells were classified according to Pedersen and Peters (1968). The small, medium and large follicular cells corresponded to type 1-3a, 3b-5a, and 5b-8 of their classification, respectively. In addition, only type 7-8 follicular cells were counted. The sections were counted twice, and the average of the two counts was calculated and reported.

Testes histology

Testes from all PND 23 male pups were fixed in Bouin’s fixative overnight at 4 °C. Samples were then dehydrated and embedded in paraffin according to standard procedures and cut into 5-μm sections. The sections were stained with hematoxylin and eosin as mentioned above. All images were captured using the same exposure time with a Zeiss AxioVision microscope and software (Carl Zeiss Microscopy, LLC). For counting the number of spermatocytes per seminiferous tubule, a total of 40 seminiferous tubules from one or two male pups from each of 3 or 4 litters/group were selected at random based on the round shape of seminiferous tubules and stage VII-IX of spermatogenesis.

Enumeration of Sertoli cells

To count Sertoli cells, we performed immunohistochemistry with an antibody to androgen receptor (AR) (Pelletier et al., 2000; Hutchison et al., 2008). In brief, slides of PND 23 male testes were deparaffinized and reacted with anti-AR antibody (1:100 dilution; Catalog #: sc-816; Santa Cruz Laboratories, Santa Cruz, CA, USA) and Elite ABC kit for rabbit IgG (Vector Laboratories, Burlingame, CA, USA). The peroxidase-labeled secondary antibody was detected using 3,3′-diaminobenzidine tetrahydrochloride (Acros Organics USA, Morris Plains, NJ, USA) as the chromogen. After immunohistochemistry, light microscopy was used to count the number of Sertoli cells per seminiferous tubule. Stage VII-IX of spermatogenesis in rat testes was selected for counting the number of Sertoli cells. Only round seminiferous tubules were selected for evaluation. Ten seminiferous tubules from one or two male pups from each of 3 litters/treatment group were selected.

Detection of apoptotic cells in testis sections

The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) method was performed using an in situ cell death detection kit (Roche Diagnostics Corp., Indianapolis, IN, USA) according to the manufacturer’s instructions. In brief, sections of PND 23 testes were treated with 10 μg/ml proteinase K in phosphate-buffered saline (PBS) at 37°C for 30 min, washed with PBS and incubated with TUNEL reaction mixture at 37°C for 60 min. After incubation, the sections were washed with PBS and counter-stained with 1 μg/ml propidium iodide (MP Biomedicals, Solon, OH, USA). Images were recorded using the same exposure time with a Zeiss AxioVision microscope and software (Carl Zeiss Microscopy, LLC). The number of seminiferous tubules with fluorescent signals (apoptotic cells) was counted using four images captured under lower magnification at random from each sample. The criterion for quantifying the fluorescent signals was 0, 1-2 and 3 or more signals per tubule (Blanchard et al., 1996). Only round seminiferous tubules of a similar size were selected. This procedure was performed on one animal from each of 3 or 4 litters/treatment group. The results were quantified by calculating the percentage of seminiferous tubules with apoptotic cells.

Measurement of serum hormone levels

Serum testosterone and thyroid-stimulating hormone (TSH) levels were measured using a microplate reader (Spectra Max 190) and accompanying software (SoftMax Pro 4.3.1 L, both from Molecular Devices, LLC, Sunnyvale, CA, USA), with testosterone ELISA, thyroid-stimulating hormone (rat) ELISA, or T3 (Triiodothyronine) (Total) ELISA kits (ALPCO Diagnostics, Salem, NH, USA). There was not sufficient blood to do both assays on each animal. If more than one animal in a litter was assayed, the average of all animals assayed in that litter is shown.

Statistics

Statistical analysis was conducted based on the number of litters per treatment group in this study. Body and organ weights shown represent mean values of all female or male pups in the litter. All other assays, except hormone assays, were done on one pup or were the mean of two pups in the litter. Group values for dam data are presented as mean ± SD. Group values of pup data are presented as mean ± SEM. Statistical analyses were performed using one-way ANOVA followed by Dunnett’s test. A p-value of ≤ 0.05 was considered statistically significant.

Results

1. Dams

Effect of HMB on body and organs weights of dams

The oral route was selected for this study for several reasons. Dermal studies in reproduction studies are complicated by grooming behaviors in which there will be both oral and dermal exposure. Additionally, several studies have demonstrated the presence of HMB in wastewater (Rodil et al., 2008; Balmer et al., 2005) and in lake water (Poiger et al., 2004). All dams survived dietary HMB exposure until the end of study; no in-life observations or clinical signs were noted that were considered related to exposure. Body weights of dams at GD 10, 15, and 20 decreased in a dose-dependent manner with a significant difference apparent in the highest dose group as early as GD 10 (Fig. 1). At GD 15, body weights of dams were also significantly reduced in the 25,000 ppm dose group (Fig. 1).

Fig. 1. Body weights of dams at GD 3-20.

Fig. 1

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Values are expressed as mean. *p ≤ 0.05 compared to the control (0 ppm) group. **p ≤ 0.01 compared to the control (0 ppm) group. Table shows number of animals per dose group at each gestational day.

Although the body weights of dams euthanized at PND 23 were not significantly different, absolute and relative liver weights were significantly increased in the 10,000 ppm and higher dose groups compared to the control group (Table 1). Absolute and relative kidney weights were also significantly higher in the highest treatment group relative to control. Paired ovaries, uterine, and relative paired ovaries and uterine weights were significantly higher in the 1,000 ppm HMB group (Table 1); however, no dose-dependent effect was noted due to the lack of effects at higher doses.

Table 1.

Necropsy body and organ weights of dams at PND 23

Control
0 ppm		 HMB dose group (ppm)
1,000 3,000 10,000 25,000 50,000
Body weights (g) 200.1 ± 22.7 214.9 ± 27.0 200.0 ± 15.4 213.8 ± 23.7 214.6 ± 26.4 204.8 ± 30.7
Liver weight (g) 6.73 ± 2.21 8.78 ± 2.19 8.12 ± 2.37 10.84 ± 2.64* 12.85 ± 2.89** 13.47 ± 2.48**
Relative liver weight (g/BW 10g) 0.330 ± 0.073 0.404 ± 0.066 0.401 ± 0.087 0.500 ± 0.080** 0.593 ± 0.069** 0.655 ± 0.038**
Kidney weight (g) 1.47 ± 0.10 1.51 ± 0.11 1.46 ± 0.13 1.52 ± 0.13 1.67 ± 0.21 1.84 ± 0.24**
Relative kidney weight (g/BW 10g) 0.074 ± 0.004 0.071 ± 0.007 0.073 ± 0.008 0.072 ± 0.005 0.078 ± 0.011 0.091 ± 0.017*
Paired ovarian weights (g) 0.086 ± 0.023 0.129 ± 0.034* 0.115 ± 0.022 0.116 ± 0.008 0.107 ± 0.011 0.091 ± 0.023
Relative paired ovaries weights (g/BW 10g) 0.0043 ± 0.0011 0.0061 ± 0.0019* 0.0057 ± 0.0010 0.0054 ± 0.0004 0.0051 ± 0.0009 0.0045 ± 0.0011
Uterine weight (g) 0.36 ± 0.13 0.61 ± 0.14** 0.40 ± 0.07 0.38 ± 0.15 0.32 ± 0.04 0.35 ± 0.09
Relative uterine weight (g/BW 10g) 0.018 ± 0.005 0.029 ± 0.007** 0.020 ± 0.002 0.018 ± 0.005 0.015 ± 0.002 0.017 ± 0.003
N 6 5 7 8 7 5a
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Values are mean ± SD excluding non-pregnant and non-nursing females.

*

p ≤ 0.05 compared to the control (0 ppm) group.

**

p ≤ 0.01 compared to the control (0 ppm) group. N indicates number of animals per dose group.

a

One animal’s data was missing of organ weights.

At GD 10-13, there was a significant decrease in food consumption in the two highest treatment groups, but this decrease was not observed at other time points (Table 2). In the postnatal period, the amount of food consumed was significantly decreased in the highest treatment group at PND 14-23, while no significant differences were observed at PND 0-7 or 7-14 (Table 2).

Table 2.

Food (g/day/animal) and estimated dose (mg/kg body weight/animal)a consumptions of dams during perinatal and postnatal period

Control
0 ppm		 HMB dose group (ppm)
1,000 3,000 10,000 25,000 50,000
Perinatal period
GD 3-6 Food 20.2 ± 3.7 (27) 20.1 ± 2.8 (26) 19.0 ± 2.4 (29) 19.4 ± 4.4 (27) 20.4 ± 3.5 (28) 20.3 ± 3.4 (30)
GD 6-10 Food 16.4 ± 2.8 (10) 15.9 ± 1.7 (8) 16.3 ± 1.5 (9) 13.8 ± 3.3 (7) 18.2 ± 10.1 (9) 14.1 ± 8.8 (8)
Estimated dose 0.0 74.9 ± 16.4 223.2 ± 35.0 712.5 ± 200.5 2041.8 ± 212.0 3749.3 ± 876.9
GD 10-13 Food 19.5 ± 2.1 (21) 17.7 ± 1.8 (18) 17.7 ± 1.8 (22) 17.1 ± 6.3 (20) 16.0 ± 3.0* (20) 15.3 ± 5.1** (22)
Estimated dose 0.0 65.7 ± 3.4 203.1 ± 6.6 626.8 ± 79.3 1747.5 ± 204.1 3214.7 ± 121.0
GD 13-20 Food 21.0 ± 3.2 (14) 20.0 ± 1.2 (11) 19.4 ± 2.2 (15) 21.0 ± 3.3 (15) 20.2 ± 1.8 (14) 19.9 ± 2.5 (15)
Estimated dose 0.0 63.2 ± 6.9 195.0 ± 18.1 673.2 ± 13.7 1605.5 ± 3.2 3380.6 ± 113.6
Postnatal period
PND 0-7 Food 21.2 ± 1.4 (6) 20.0 ± 10.4 (6) 26.2 ± 10.4 (7) 21.3 ± 1.6 (8) 19.9 ± 3.3 (7) 17.5 ± 3.1 (82.2)
Estimated dose 0.0 66.6 ± 11.7 249.5 ± 95.2 756.2 ± 131.1 1864.7 ± 363.3 3756.8 ± 418.6
PND 7-14 Food 35.4 ± 13.0 (6) 27.7 ± 7.1 (6) 38.7 ± 7.9 (7) 36.7 ± 8.9 (8) 32.1 ± 4.6 (7) 28.1 ± 2.2 (6)
Estimated dose 0.0 88.4 ± 19.0 393.0 ± 107.8 1173.8 ±459.4 2793.3 ± 950.0 5274.2 ± 1727.3
PND 14-23 Food 67.1 ± 17.4 (6) 62.0 ± 21.0 (6) 56.2 ± 7.8 (7) 64.5 ± 19.2 (8) 50.1 ± 13.0 (7) 32.2 ± 7.4 * (4)
Estimated dose 0.0 195.1 ± 132.0 656.1 ± 264.3 2257.7 ± 1073.6 4650.7 ± 1676.8 7178.5 ± 965.7
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Values are mean ± SD excluding non-pregnant females. ( ) indicates number of animals per dose group

a

Estimated dose consumption (mg/kg body weight)= mean food consumption (kg) × dose (mg/kg chow)/mean body weight (kg) for each time point. Estimated dose during a time period (GD 6-10, GD 10-13, GD 13-20, PND 0-7, PND7-14 and PND 14-23) was averaged over those time points.

*

p ≤ 0.05 compared to the control (0 ppm) group.

**

p ≤ 0.01 compared to the control (0 ppm) group.

HMB and metabolite concentrations in dams

HMB and metabolite concentrations in serum from dams are shown in Table 3. HMB and DHB levels in the dose groups increased in a dose-dependent manner at GD 10, 15, 20 and PND 23. THB and DHMB metabolite levels were not detected in this study. Systemic levels of HMB were also under the LOD for the 1,000 ppm dose group at all time points prior to PND 23. Surprisingly, at PND 23, there were very low levels of HMB detected in the control group as well. It is unclear why this occurred, but environmental and/or husbandry contamination cannot be ruled out.

Table 3.

HMB and metabolites concentrations (μg/mL) in serum of dams on GD 10, GD 15, GD 20 and PND 23

Control HMB dose group (ppm)
0 ppm 1,000 3,000 10,000 25,000 50,000
HMB DHB HMB DHB HMB DHB HMB DHB HMB DHB HMB DHB
GD 10 <LOD <LOD <LOD 0.0422 ± 0.0138 0.0174 ± 0.0062 0.2406 ± 0.3618 0.0312 ± 0.0126 0.1652 ± 0.0459 0.1250 ± 0.0294 0.2903 ± 0.0830 0.1886 ± 0.1019 0.3097 ± 0.1118
N 6 6 6 6 6 6
GD 15 <LOD <LOD <LOD 0.0425 ± 0.0137 0.0062 ± 0.001 0.0571 ± 0.0171 0.0481 ± 0.0151 0.2155 ± 0.0392 0.1385 ± 0.0161 0.3070 ± 0.0359 0.2318 ± 0.1070 0.4012 ± 0.1401
N 6 6 6 6 6 6
GD 20 <LOD <LOD <LOD 0.0307 ± 0.0187 0.0096 ± 0.0009 0.0812 ± 0.0314 0.0660 ± 0.0417 0.1774 ± 0.0862 0.1466 ± 0.0721 0.3147 ± 0.1866 0.4850 ± 0.2496 0.6215 ± 0.2762
N 6 5 6 6 6 6
PND 23 0.0118 ± 0.0070 <LOD 0.0072 ± 0.0008 0.0382 ± 0.0122 0.0164 ± 0.0139 0.1284 ± 0.0423 0.1011 ± 0.0310 0.2562 ± 0.0599 0.4804 ± 0.0899 0.7748 ± 0.1397 0.6886 ± 0.2447 1.0066 ± 0.3874
N 5 5 5 5 5 5
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Values are mean ± SD. N indicates number of litter per dose group. LOD of HMB, DHB and DHMB is < 0.005 μg/ml. LOD of THB is < 0.1 μg/ml (GD 10, 15 and PND 23) or 0.05 μg/ml (GD 20). THB and DHMB concentrations were under the limit of detection (LOD) in all analyses.

Effect of HMB on clinical chemistry of dams

ALT levels were significantly increased in the 25,000, and 50,000 ppm HMB groups at GD 10, 15, 20 and PND 23 (Table 4). ALP was elevated at the three highest doses at all time points. Total bile acids were elevated at the two highest doses at GD 10, 15 and PND 23, but only at the highest dose at GD 20. Creatinine was significantly decreased at the highest dose on GD 15 and 20 and at the two highest doses on PND 23. Additionally, at PND 23, glucose, total protein, albumin and cholesterol levels were significantly elevated in the 50,000 ppm HMB group, while BUN and AST levels were significantly lower in the 50,000 ppm HMB group (Table 4).

Table 4.

Clinical chemistry analysis in serum of dams sacrificed at GD 10, 15, 20 and PND 23

Control
0 ppm		 HMB dose group (ppm)
1,000 3,000 10,000 25,000 50,000
Glucose (mg/dL) GD 10 129.2 ± 5.5 131.6 ± 16.4 139.3 ± 14.0 132.9 ± 12.4 132.3 ± 18.7 117.3 ± 9.4
GD 15 103.9 ± 13.0 105.0 ± 0.5 112.1 ± 20.2 109.0 ± 7.3 117.8 ± 18.6 117.0 ± 16.9
GD 20 67.4 ± 16.1 78.6 ± 24.2 72.0 ± 19.5 99.7 ± 30.7* 86.3 ± 18.9 88.4 ± 13.6
PND 23 87.8 ± 17.7 100.6 ± 23.2 111.9 ± 12.2 111.8 ± 19.7 136.9 ± 15.8** 143.2 ± 10.9**
Total protein (g/dL) GD 10 6.5 ± 0.3 6.4 ± 0.5 6.5 ± 0.3 6.3 ± 0.4 6.0 ± 0.5 6.3 ± 0.4
GD 15 6.3 ± 0.4 6.4 ± 0.5 6.4 ± 0.3 7.0 ± 0.4 7.0 ± 0.4 6.8 ± 0.4
GD 20 5.2 ± 0.7 5.5 ± 0.3 5.4 ± 0.3 5.8 ± 0.6 6.3 ± 0.7** 5.7 ± 0.3
PND 23 6.0 ± 0.5 5.8 ± 0.4 6.2 ± 0.5 6.0 ± 0.4 6.1 ± 0.2 6.9 ± 0.8*
Albumin (g/dL) GD 10 3.5 ± 0.2 3.5 ± 0.2 3.6 ± 0.1 3.5 ± 0.2 4.0 ± 0.2 3.6 ± 0.2
GD 15 3.6 ± 0.2 3.7 ± 0.2 3.7 ± 0.1 3.9 ± 0.2 4.0 ± 0.2 4.0 ± 0.2**
GD 20 2.7 ± 0.3 2.8 ± 0.1 2.8 ± 0.2 3.0 ± 0.3 3.4 ± 0.4** 3.0 ± 0.1
PND 23 3.2 ± 0.2 3.1 ± 0.5 3.2 ± 0.3 3.0 ± 0.2 3.5 ± 0.1 3.9 ± 0.4*
ALT (U/L) GD 10 67.0 ± 10.9 67.5 ± 8.8 69.1 ± 7.3 79.4 ± 15.2 106.0 ± 19.0** 110.0 ± 27.1**
GD 15 68.6 ± 9.8 70.7 ± 6.9 67.0 ± 15.9 86.5 ± 7.3 111.0 ± 22.1** 124.0 ± 30.1***
GD 20 63.9 ± 8.7 62.4 ± 4.9 64.5 ± 11.3 63.7 ± 18.5 94.0 ± 30.5* 111.0 ±16.9***
PND 23 148.3 ± 45.0 164.2 ± 35.5 176.0 ± 38.1 231 ± 64.7* 270.0 ± 52.1** 241.0 ± 66.1*
AST (U/L) GD 10 77.3 ± 5.1 80.9 ± 7.5 81.7 ± 4.5 92.0 ± 16.8 93.0 ± 16.7 92.0 ± 10.2
GD 15 77.7 ± 10.1 77.3 ± 6.3 83.7 ± 13.5 81.5 ± 4.9 86.0 ± 13.7 85.0 ± 10.5
GD 20 79.4 ± 30.4 76.8 ± 18.9 70.6 ± 10.1 134.3 ± 95.1 67.0 ± 11.3 66 ± 10.9
PND 23 160.0 ± 22.8 158.4 ± 28.8 157.1 ± 19.9 158 ± 20.3 134.0 ± 18.6 109.0 ± 17.7**
Cholesterol (mg/dL) GD 10 99.2 ± 8.8 99.3 ± 13.0 108.3 ± 11.6 105.3 ± 6.9 107.0 ± 19.8 102.0 ± 20.0
GD 15 82.1 ± 8.8 87.0 ± 12.7 87.6 ± 10.0 76.5 ± 13.0 73.0 ± 7.3 61.0 ± 17.1**
GD 20 112.0 ± 10.1 118.0 ± 12.9 123.3 ± 8.9 124.9 ± 20.4 108.0 ± 14.5 100.0 ± 19.2
PND 23 67.5 ± 24.8 83.6 ± 43.9 65.6 ± 21.7 91 ± 35.6 88.0 ± 26.8 114.0 ± 27.0*
Triglyceride (mg/dL) GD 10 75.7 ± 23.1 83.1 ± 20.2 72.0 ± 13.9 67.9 ± 14.5 92.0 ± 23.3 71.0 ± 22.8
GD 15 113.9 ± 25.0 145.4 ± 51.3 94.9 ± 27.8 143.0 ± 49.0 84.0 ± 8.3 82.0 ± 24.6
GD 20 344.0 ± 106.8 355.2 ± 193.6 289.9 ± 90.6 261.6 ± 110.9 313.0 ± 122.1 472.0 ± 92.2
PND 23 34.8 ± 21.2 46.4 ± 18.6 35.1 ± 18.6 32 ± 11.5 26.0 ± 15.0 36.0 ± 32.5
BUN (mg/dL) GD 10 20.8 ± 2.8 19.6 ± 3.5 20.3 ± 3.5 19.6 ± 2.8 19.0 ± 2.9 17.0 ± 3.4
GD 15 18.3 ± 3.3 20.7 ± 1.5 17.6 ± 2.9 20.0 ± 3.6 23.0 ± 3.5 20.0 ± 4.9
GD 20 17.9 ± 2.1 18.0 ± 1.9 17.4 ± 1.7 17.6 ± 3.1 20.0 ± 4.5 19.0 ± 1.2
PND 23 58.0 ± 21.6 40.6 ± 12.2 41.6 ± 11.3 45.0 ± 15.6 38.0 ± 6.8 36.0 ± 11.3*
Creatinine (mg/dL) GD 10 0.5 ± 0.1 0.5 ± 0.1 0.5 ± 0.1 0.4 ± 0.1 0.5 ± 0.1 0.4 ± 0.1
GD 15 0.5 ± 0.1 0.5 ± 0.0 0.4 ± 0.1 0.4 ± 0.1 0.4 ± 0.1 0.4 ± 0.1*
GD 20 0.5 ± 0.0 0.5 ± 0.1 0.5 ± 0.1 0.5 ± 0.1 0.4 ± 0.1 0.3 ± 0.1*
PND 23 0.6 ± 0.1 0.6 ± 0.1 0.6 ± 0.1 1.0 ± 0.1 0.4 ± 0.1** 0.4 ± 0.1*
ALP (U/L) GD 10 198.0 ± 22.9 192.3 ± 33.4 223.0 ± 54.0 270.1 ± 61.2* 308.0 ± 25.4** 268.0 ± 58.9*
GD 15 164.7 ± 24.6 190.9 ± 18.1 158.3 ± 28.3 234.3 ± 34.9* 301.0 ± 44.4*** 305.0 ± 82.3**
GD 20 94.7 ± 14.0 109.0 ± 25.2 101.6 ± 24.8 164.6 ± 50.8* 168.0 ± 54.8* 211.0 ± 59.8***
PND 23 262.0 ±182.7 291.4 ± 130.4 452.4 ± 290.2 658.0 ± 306.8* 846 ± 269.8** 772 ± 327.0*
Total bile acids (TBA) (μmol/L) GD 10 53.9 ± 33.6 63.3 ± 21.6 57.9 ± 29.4 11.97 ± 37.5 164.0 ± 71.0** 142.0 ± 83.1*
GD 15 63.4 ± 33.2 62.1 ± 32.9 67.4 ± 25.7 91.4 ± 43.0 165.0 ± 64.6* 207.0 ± 106.6***
GD 20 60.0 ± 32.0 79.8 ± 61.3 71.3 ± 42.4 104.1 ± 109.5 102.0 ± 45.4 161.0 ± 49.2*
PND 23 111.4 ± 125.2 93.2 ± 50.9 60.0 ± 14.9 130.0 ± 72.4 329.0 ± 137.5** 380.0 ± 147.2***
Inorganic (I) phosphorus (mg/dL) GD 10 6.9 ± 0.7 7.3 ± 0.8 7.5 ± 0.7 7.8 ± 1.0 8.0 ± 1.4 8.0 ± 1.0
GD 15 7.5 ± 0.9 7.7 ± 0.7 8.0 ± 1.0 8.2 ± 0.8 8.0 ± 1.0 8.0 ± 0.9
GD 20 7.4 ± 0.5 7.3 ± 0.7 7.6 ± 0.6 8.1 ± 1.4 7.0 ± 0.8 8.0 ± 1.1
PND 23 9.9 ± 0.8 9.4 ± 0.9 8.5 ± 2.1 9.0 ± 1.7 10.0 ± 1.1 10.0 ± 1.6
Calcium (mg/dL) GD 10 10.3 ± 0.5 10.6 ± 0.2 10.4 ± 0.7 10.4 ± 0.4 11.0 ± 0.6 11.0 ± 0.7
GD 15 10.8 ± 0.6 10.8 ± 0.6 10.8 ± 0.6 10.8 ± 0.6 11.0 ± 0.2 11.0 ± 0.5
GD 20 9.8 ± 0.4 10.2 ± 0.3 10.2 ± 0.4 10.3 ± 0.5 11.0 ± 0.7 11.0 ± 0.5
PND 23 9.7 ± 0.8 10.1 ± 0.9 9.8 ± 0.9 10.0 ± 0.6 11.0 ± 0.5 11.0 ± 0.5
Sorbitol dehydrogenase (U/L) GD 10 18.0 ± 6.7 21.8 ± 11.0 22.5 ± 7.2 19.5 ± 6.5 25.0 ± 11.6 23.0 ± 4.0
GD 15 20.8 ± 8.6 25.6 ± 13.6 27.3 ± 14.2 23.9 ± 15.1 25.0 ± 17.5 22.0 ± 13.8
GD 20 25.9 ± 11.8 26.9 ± 8.9 33.7 ± 11.1 26.6 ± 13.3 28.0 ± 17.9 22.0 ± 7.1
PND 23 34.0 ± 10.8 20.7 ± 13.4 30.2 ± 13.6 27.0 ± 6.8 22.0 ± 10.0 19.0 ± 7.1
N GD 10 6 8 7 7 8 7
GD 15 7 7 7 6 6 8
GD 20 7 5 8 7 7 8
PND 23 6 5 7 8 7 6
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Values are mean ± SD excluding non-pregnant animals. GD: gestation day; N: samples number.

*

p ≤ 0.05 compared to the control (0 ppm) group.

**

p ≤ 0.01 compared to the control (0 ppm) group.

***

p ≤ 0.005 compared to the control (0 ppm) group.

Effect of HMB on reproductive/pregnancy parameters

No significant differences were observed in the mean number of implantation sites/litter, mean resorptions/litter and % litters with resorptions between the control and the HMB dose groups at GD 10, 15, and 20 (Supplemental Table 1). There was also no significant difference in the number of litters with surviving pups at PND 23 (Supplemental Table 1). Moreover, there were no significant differences in the number or weights of live fetuses (total, male or female), and no difference in the sex ratios among the control and all dose groups at GD 20 (Supplemental Table 2). One fetus in the 50,000 ppm HMB group had hydrocephaly, but no other malformations were observed.

2. Offspring

2.1. Body weight gain of offspring from PND 1-23 and clinical chemistry in offspring at PND 23

Initially there were no differences in body weights of either male or female pups treated with HMB, but by PND 14 weights were decreased at the highest dose group for both sexes (Fig. 2). In addition, body weights of male and female pups were significantly less than control pups at PND 23 in the 25,000 ppm group (Fig. 2). Although no significant differences in body weight adjusted AGD were observed at PND 1 (Fig. 3, left panel), there was a significant reduction in normalized AGD in 50,000 ppm HMB dose group of PND 23 male pups (Fig. 3, right panel). No differences in nipple retention and testes descent were observed in any of the dose groups (data not shown).

Fig.2. Average Body weights of male (left) and female (right) offspring at PND 1-23.

Fig.2

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Values are expressed as mean of all litters; all pups of one sex were included for each litter mean. *p ≤ 0.05 compared to the control (0 ppm) group. **p ≤ 0.01 compared to the control (0 ppm) group. Table shows number of litters per dose group at each postnatal day.

Fig. 3. AGD normalized to body weight in male and female offspring at PND 1 (left) and 23 (right).

Fig. 3

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Values are expressed as mean ± SEM. N indicates number of litters per dose group; all pups of each sex within a litter were included in the analyses. *p ≤ 0.05 compared to the control (0 ppm) group.

At PND 23, serum ALT was decreased at the two highest doses in male pups; ALT was decreased in female pups only in the 25,000 ppm group. Cholesterol levels in both female and male offspring were significantly increased in the 25,000 and 50,000 ppm HMB dose groups (Table 5).

Table 5.

Clinical chemistry analysis in serum of female and male offspring

Control
0 ppm		 HMB dose group (ppm)
1,000 3,000 10,000 25,000 50,000
Male ALT (U/L) 49.8 ± 9.8 51.4 ± 5.2 48.7 ± 7.8 47.5 ± 10.7 34.6 ± 4.9* 24.0 ± 3.7**
Cholesterol (mg/dL) 146.8 ± 3.7 152.4 ± 19.0 199.2 ± 47.2 211.3 ± 54.2 332.6 ± 50.7** 470.0 ± 93.5***
Creatinine (mg/dL) 0.3 ± 0.0 0.3 ± 0.0 0.3 ± 0.1 0.3 ± 0.0 0.2 ± 0.1 0.2 ± 0.0*
Glucose (mg/dL) 130.8 ± 19.2 114.8 ± 27.3 125.2 ± 14.6 147.6 ± 55.3 129.6 ± 6.0 140.7 ± 34.9
Total protein (g/dL) 5.1 ± 0.2 5.1 ± 0.3 5.3 ± 0.2 5.4 ± 0.6 5.2 0.2 4.8 ± 0.2
ALP (U/L) 276.4 ± 76.6 338.4 ± 96.2 357.5 ± 52.8 376.3 ± 172.8 327.6 ± 50.8 327.3 ± 19.2
AST (U/L) 127.5 ± 9.8 121.4 ± 13.2 137.5 ± 16.3 145.3 ± 56.6 133.2 ± 18.6 119.3 ± 10.5
Albumin (g/dL) 3.2 ± 0.2 3.1 ± 0.1 3.2 ± 0.3 3.1 ± 0.3 3.2 ± 0.2 3.0 ± 0.0
I phosphorus (mg/dL) 11.3 ± 1.1 11.1 ± 0.4 11.9 ± 2.6 11.2 ± 0.6 10.3 ± 0.6 11.7 ± 1.2
SDH (U/L) 11.1 ± 11.3 11.0 ± 9.3 11.0 ± 12.2 3.4 ± 5.1 1.4 ± 2.5 17.4 ± 24.6
BUN (mg/dL) 27.8 ± 9.4 18.6 ± 3.0 23.0 ± 4.2 26.4 ± 5.3 28.8 ± 4.8 29.0 ± 5.0
Calcium (mg/dL) 11.0 ± 0.7 11.2 ± 0.7 11.5 ± 0.5 11.4 ± 0.8 11.2 ± 0.2 10.9 ± 0.2
TBA (μmol/L) 101.9 ± 42.4 72.4 ± 27.7 104.8 ± 36.6 116.7 ± 30.8 96.6 ± 23.9 117.6 ± 33.8
N 4-5 5 6 8 5 2-3a
Female ALT (U/L) 54.0 ± 10.9 50.6 ± 14.6 47.1 ± 11.7 46.8 ± 6.7 35.2 ± 4.2* 34.5 ± 5.5
Cholesterol (mg/dL) 158.7 ± 26.5 159.4 ± 8.8 166.7 ± 24.7 225.8 ± 69.8 288.6 ± 73.5** 410.5 ± 53.5**
Creatinine (mg/dL) 0.3 ± 0.1 0.2 ± 0.1 0.3 ± 0.0 0.2 ± 0.1 0.2 ± 0.1 0.2 ± 0.0
Glucose (mg/dL) 128.5 ± 12.9 123.8 ± 25.0 126.1 ± 10.8 138.1 ± 27.9 135.0 ± 7.7 123.0 ± 26.0
Total protein (g/dL) 5.2 ± 0.2 5.3 ± 0.2 5.2 ± 0.2 5.3 ± 0.2 5.4 ± 0.1 5.4 ± 0.1
ALP (U/L) 291.0 ± 110.5 350.6 ± 124.3 353.1 ± 127.4 376.1 ± 126.4 296.8 ± 57.4 406.5 ± 76.5
AST (U/L) 136.3 ± 22.4 148.8 ± 28.2 133.4 ± 12.0 130.4 ± 10.8 123.4 ± 10.7 103.0 ± 22.0
Albumin (g/dL) 3.2 ± 0.2 3.2 ± 0.1 3.1 ± 0.1 3.1 ± 0.1 3.2 ± 0.1 3.2 ± 0.1
I phosphorus (mg/dL) 11.7 ± 1.2 11.7 ± 0.9 10.6 ± 0.7 11.4 ± 0.8 10.9 ± 0.4 12.0 ± 0.3
SDH (U/L) 7.5 ± 5.8 5.7 ± 7.6 8.5 ± 6.1 3.6 ± 6.1 1.1 ± 2.2 0.0 ± 0.0
BUN (mg/dL) 29.7 ± 9.4 21.4 ± 3.3 22.4 ± 5.0 26.0 ± 3.2 34.6 ± 3.6 38.0 ± 0.0
Calcium (mg/dL) 11.3 ± 0.3 11.1 ± 0.8 11.1 ± 0.4 11.5 ± 0.8 11.7 ± 0.8 12.0 ± 0.4
TBA (μmol/L) 136.8 ± 44.3 159.9 ± 48.2 107.5 ± 41.6 128.0 ± 54.3 118.0 ±55.1 84.8 ± 0.0
N 6 7 7 8 5 1-2a
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Values are mean ± SD. N: samples number.

a

Two-three litters could not collect blood due to small body size.

*

p ≤ 0.05 compared to the control (0 ppm) group.

**

p ≤ 0.01 compared to the control (0 ppm) group.

***

p ≤ 0.005 compared to the control (0 ppm) group.

2.2. Female offspring

Effect of HMB on female offspring body and organ weights at PND 23

As shown in Table 6, although body and paired ovarian weights on PND 23 were significantly decreased at the two highest dose groups, ovarian/body weight ratios were not different among the groups. Uterine weight was also significantly lower in the group treated with 50,000 ppm HMB; however, the uterine/body weight ratios were not significantly different. Relative liver to body weight ratios were significantly increased in the 10,000, 25,000 and 50,000 ppm HMB groups. Paired kidney weights were significantly lower than the control group; however, relative paired kidney weights to body weight ratios were not significantly different from controls.

Table 6.

Body and organ weights in PND 23 female offspring

Control
0 ppm		 HMB dose group (ppm)
1,000 3,000 10,000 25,000 50,000
Body weight (BW)(g) 35.74 ± 2.86 39.89 ± 2.00 34.06 ± 2.13 34.64 ± 2.06 27.81 ± 1.32* 22.53 ± 2.93*
Paired ovarian weights (g) 0.023 ± 0.002 0.026 ± 0.002 0.023 ± 0.002 0.021 ± 0.001 0.017 ± 0.001* 0.014 ± 0.002*
Relative paired ovarian weights/BW (g/10g BW) 0.007 ± 0.0003 0.007 ± 0.0003 0.007 ± 0.0002 0.006 ± 0.0002 0.006± 0.0002 0.006 ± 0.0003
Uterine weights (g) 0.040 ± 0.006 0.041 ± 0.005 0.035 ± 0.002 0.035 ± 0.004 0.029 ± 0.003 0.023 ± 0.002*
Relative uterine weights/BW (g/10g BW) 0.011 ± 0.001 0.010± 0.001 0.012 ± 0.0004 0.010 ± 0.0006 0.010 ± 0.0005 0.010 ± 0.0007
Liver weight (g) 1.22 ± 0.11 1.49 ± 0.15 1.31 ± 0.13 1.46 ± 0.10 1.19 ± 0.05 0.92 ± 0.14
Relative liver weight/BW (g/10g BW) 0.34 ± 0.01 0.37 ± 0.02 0.38 ± 0.02 0.42 ± 0.01** 0.43 ± 0.01** 0.41 ± 0.01*
Paired kidney weights (g) 0.36 ± 0.03 0.41 ± 0.02 0.36 ± 0.02 0.37 ± 0.02 0.31 ± 0.02 0.25 ± 0.03*
Relative paired kidney weights/BW (g/10g BW) 0.103 ± 0.001 0.102 ± 0.001 0.106 ± 0.004 0.106 ± 0.002 0.112 ± 0.003 0.112 ± 0.002
N 6 5 6 8 7 4
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Values are mean ± SEM of all female pups in the litter.

*

p ≤ 0.05 compared to the control (0 ppm) group.

**

p ≤ 0.01 compared to the control (0 ppm) group.

N indicates number of animals per dose group.

Effect of HMB on ovarian histology in PND 23 female offspring

Follicular development in ovaries of control and all HMB dose groups is shown in Fig. 4. There were no differences in the number of small, medium, or large follicles in any dose group when compared to the control group (Supplemental Table 3). However, the number of antral follicles (type 7-8, arrows in Fig. 4) was significantly lower in the 10,000 and 50,000 ppm HMB dose groups (Supplemental Table 3).

Fig.4. Histology of PND 23 juvenile ovaries.

Fig.4

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Ovary sections from female offspring were stained by hematoxylin and eosin. Shown are control and all HMB dose groups. Arrows indicated type 7-8 follicular cells. Bar: 50 μm.

2.3. Male offspring

Effect of HMB on male offspring body and organ weights at PND 23

As shown in Table 7, body and paired testes weights on PND 23 decreased in a dose-dependent manner as a result of HMB exposure with a significant difference being apparent in the highest dose group; however, the testes/body weight ratios were not different among the groups (Table 7). Paired epididymides weights were also lower in the groups treated with 25,000 and 50,000 ppm HMB; however, these decreases were not statistically different when compared to the control group. Absolute and relative liver weights were not significantly different. Paired kidney weights in the highest group were significantly decreased; however, there were no differences in relative paired kidney weights to body weight ratios among the control and dose groups.

Table 7.

Body and organ weights in PND 23 male offspring

Control
0 ppm		 HMB dose group (ppm)
1,000 3,000 10,000 25,000 50,000
Body weight (BW)(g) 37.66 ± 3.28 41.79 ± 2.05 35.39 ± 1.91 35.67± 2.46 29.58 ± 1.13* 24.8 ± 3.68*
Paired testes weights (g) 0.24 ± 0.02 0.28 ± 0.02 0.23 ± 0.01 0.23 ± 0.02 0.19 ± 0.01 0.16 ± 0.03*
Relative paired testes weights/BW (g/10g BW) 0.063 ± 0.001 0.067 ± 0.003 0.069 ± 0.006 0.065 ± 0.002 0.064 ± 0.001 0.064 ± 0.003
Paired epididymides weights (g) 0.036 ± 0.002 0.039 ± 0.002 0.038 ± 0.003 0.040 ± 0.003 0.028 ± 0.001 0.026 ± 0.001
Relative paired epididymides weights/BW (g/10g BW) 0.010 ± 0.001 0.009 ± 0.001 0.012 ± 0.002 0.011 ± 0.001 0.009 ± 0.0003 0.011 ± 0.0009
Liver weight (g) 1.32 ± 0.15 1.57 ± 0.15 1.43 ± 0.17 1.52 ± 0.16 1.18 ± 0.06 0.96 ± 0.17
Relative liver weight/BW (g/10g BW) 0.35 ± 0.01 0.37 ± 0.02 0.42 ± 0.03 0.42 ± 0.02 0.40 ± 0.01 0.38 ± 0.01
Paired kidney weights (g) 0.39 ± 0.04 0.42 ± 0.02 0.35 ± 0.01 0.37 ± 0.03 0.30 ± 0.01 0.25 ± 0.04*
Relative paired kidney weights/BW (g/10g BW) 0.102 ± 0.001 0.101 ± 0.001 0.107 ± 0.010 0.103 ± 0.002 0.102 ± 0.001 0.103 ± 0.001
N 6 5 7 8 7 4
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Values are mean ± SEM of all male pups in the litter.

*

p ≤ 0.05 compared to the control (0 ppm) group.

N indicates number of animals per dose group.

Effect of HMB on testis morphology of PND 23 rat offspring

As shown in Fig. 5, some seminiferous tubules in the 50,000 ppm HMB group contained a few or no spermatocytes (Fig. 5A, arrows) when compared to the control (0 ppm) group (Fig. 5A). In Stage VII-IX, the number of spermatocytes per seminiferous tubule was significantly reduced at doses of 3,000 ppm and higher (Table 8), while the number of Sertoli cells per seminiferous tubule in all dose groups was not altered (Fig. 5B; Table 8).

Fig. 5. Histology of rat testes at PND 23 in the HMB dose groups.

Fig. 5

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(A) Hematoxylin and eosin-stained testis sections of seminiferous tubules from the control and 50,000 ppm groups are shown. Different sections (middle and lower panels) are shown under higher magnification. Arrows indicate abnormal seminiferous tubules. (B) Testis sections of rat male offspring at PND 23 from the control and 50,000 ppm groups were immunostained with antibody to AR as a marker of Sertoli cells. Stage VII-IX of spermatogenesis in rat testes was selected for counting the number of Sertoli cells. Bars: 20 μm.

Table 8.

Numbers of spermatocytes and Sertoli cells per seminiferous tubule in the testes of control and HMB dose groups

Control
0 ppm		 HMB dosed groups (ppm)
1,000 3,000 10,000 25,000 50,000
Spermatocytesa 36.32 ± 2.24 31.22 ± 1.61 19.72 ± 2.53* 26.08± 1.14* 17.04 ± 1.30* 7.86 ± 1.19*
Sertoli cellsb 50.80 ± 0.98 51.53± 1.73 52.80 ± 2.41 51.73 ± 1.70 50.77 ± 1.30 52.97 ± 1.93
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a

Values are mean ± SEM of 3-4 litters per group.

b

Values are mean ± SEM of 3 litters per group.

*

p ≤ 0.05 compared to the control (0 ppm) group. These data were collected from two male pups per litter.

Abnormal germ cells or cells with densely stained nuclei were also observed in some of the dose groups (data not shown). Therefore, TUNEL staining was performed to determine whether this was associated with apoptotic cell death in the testis (Fig. 6). Many apoptotic cells were indeed present in the seminiferous tubules at HMB doses of 3,000 ppm and higher (Fig. 6A). When comparing the number of seminiferous tubules with three or more apoptotic cells (Blanchard et al., 1996), the number of tubules was significantly higher at 10,000 ppm HMB and all higher doses compared to controls (Fig. 6B).

Fig. 6. Detection of apoptotic cells in rat testes at PND 23 in HMB dose groups.

Fig. 6

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(A) Observation of TUNEL staining in rat testes at PND 23 is shown. Many TUNEL-positive cells (white spots) in the seminiferous tubules of the testes of rats treated with 3,000 ppm and higher doses of HMB were observed compared to the control (0 ppm) group. Bars: 20 μm. (B) The graph shows the percentage of seminiferous tubules with three or more TUNEL positive cells per dose group. Values are expressed as mean ± SEM of 3-4 litters per group. *p ≤ 0.05 compared to the control (0 ppm) group.

Effect of HMB on serum testosterone levels in PND 23 male offspring

To determine the effect of maternal and lactational HMB exposure on serum testosterone levels, hormone levels in PND 23 male rat offspring were measured. Serum testosterone levels were significantly lower in groups exposed to 3,000, and 25,000 ppm HMB compared to the controls (Fig. 7); however, these sporadic decreases do not appear to be treatment-related.

Fig. 7. Serum testosterone levels in PND 23 male rats.

Fig. 7

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Values are expressed as mean ± SEM of 4-7 litters per group. *p ≤ 0.05 compared to the control (0 ppm) group.

Discussion

In this study, to determine if the effects of maternal and lactational exposure to HMB on embryonic development and development of the reproductive organs in rat offspring at PND 23, time-mated female rats were fed a chow containing HMB (0, 1,000, 3,000, 10,000, 25,000, or 50,000 ppm) from GD 6 until PND 23. This study found that maternal and lactational exposure to HMB was associated with a number of significant findings at the highest or two highest dose levels (e.g., normalized AGD and decreased number of spermatocytes in males at PND 23, body and organ weights and several clinical chemistry parameters in dams and pups). Some of these differences may be secondary effects due to an HMB-induced growth delay such that postnatal development is somewhat stunted compared to normal development.

This study evaluated serum HMB and its metabolites in rats dosed with HMB in chow (Table 3). Other studies have measured HMB and metabolite levels in blood or plasma of rats given HMB by gavage (el Dareer et al., 1986; Okereke et al., 1993; Kadry et al., 1995). Okereke et al. (1993) observed 0.45 μg/mL HMB in plasma from rats 20 hours after exposure to 100 mg/kg HMB by gavage; el Dareer et al. (1986) found 0.004, 0.029, 0.266, and 7.62 μg/mL HMB in plasma from rats 72 hours after exposure to 3.01, 28.1, 293 or 2,570 mg/kg HMB by gavage, respectively. And also, el Dareer et al. (1986) demonstrated that 65-72% of the total dose of HMB was detected in urine 0-72 hours after gavage dosing.

There is one human study with dermal application that evaluated the plasma HMB concentration 0-96 hours after dosing (Janjua et al.,2008); HMB was applied in cream (2 mg/cm2 corresponding to 40 g/2.0 m2 of body area) on human skin daily for four days. Peak HMB levels (median) of 0.187 μg/mL in females and 0.238 μg/mL in males were observed in plasma 4 hours after treatment (females) and 3 hours after the treatment (males). Twenty-four hours after the first application, the median HMB levels in plasma were 0.047 μg/mL in females and 0.041 μg/mL in males (Janjua et al., 2008).

In order to compare doses between humans and animals, Reagan-Shaw et al. (2008) suggested that the human equivalent dose (HED) can be calculated using a body surface area normalization method. By multiplying the animal dose (mg/kg) by the conversion factor (Km) for rats to humans, the HED can be determined. Using only data from gestational exposure to HMB in the current study, the average consumed doses were 67.9, 207.1, 670.8, 1,798.3 and 3,448.2 mg/kg/day (averaged from data in Table 2) for the 1,000, 3,000, 10,000, 25,000 and 50,000 ppm doses, respectively. Using the formula proposed by Reagan-Shaw et al. (2008) for dose translation from animal to human studies, the HEDs from this study are 11.01, 33.59, 108.80, 296.68 and 559.30 mg/kg [HED (mg/kg) = animal dose (mg/kg) × Km factor animal/Km factor human; 6 is the Km for rats and 37 is the Km for humans]. The plasma concentration of 0.047 μg/mL observed in human females 24 hours after topical exposure of HMB (Janjua et al., 2008) is very similar to the plasma concentration of 0.048 μg/mL observed at the 10,000 ppm dose in this study. Janjua et al. (2008) applied HMB at 20 g/m2 (or 2 mg/cm2); if the ingested 10,000 ppm rat dose in our study is converted, the result is a dose of 4.02 g/m2 [0.6708 g/kg × 6 (Km factor for rat)]. Based on plasma concentrations, it appears that the 10,000 ppm dose in this study is within 5-fold of the dermal dose applied by Janjua et al. (2008). However, the concentration applied by Janjua’s group is generally considered to be excessive. Wang et al. (2011) used a dose of 1 mg/cm2 which they considered to be “a generous in-use dose.” This would suggest that based on similar plasma levels, the 10,000 ppm dose in the current study is about 2-fold higher than what might be considered a more typical human dose. All higher doses in this study would be considered to be in excess of typical human exposure.

Many of the adverse effects of HMB in the dams and offspring in the present study are due to the very high doses used. Although comparisons of the exposure levels of HMB between rats and humans are difficult, the studies mentioned above indicate that based on serum HMB levels and dose, exposure levels of the rats dosed with 3,000-10,000 ppm HMB in this study appear to be similar to the levels of HMB exposure in humans. Focusing on results in the 10,000 ppm dose group, absolute and normalized liver weight of the dams were elevated as was glucose at GD 20, ALT at PND 23 and ALP at all time points. This suggests possible hepatotoxicity (Fujii, 1997; Giboney, 2005), and these results are consistent with those reported in the earlier NTP study (National Toxicology Program, 1992). In that study, absolute and relative liver weights were significantly increased by HMB treatment in both sexes at all doses of 3,125 ppm and higher; absolute and relative kidney weights were increased only at 50,000 ppm in males and at 25,000 and 50,000 ppm in females. Inflammation of the kidney was also observed histologically at 50,000 ppm in the NTP study.

Very few effects were observed in the offspring at 10,000 ppm or lower doses. Normalized liver weight was increased in female offspring at 10,000 ppm, but this was not true for male offspring. The reason for the difference between the sexes is not clear and was not reflected in the clinical chemistry parameters. The number of spermatocytes was decreased at both 3,000 and 10,000 ppm; there was also an increase in apoptotic germ cells in offspring treated at the 10,000 ppm dose (Fig. 6; Table 8). Some chemicals with endocrine disrupting activity such as 2,3,7,8-tetracholorodibenzo-p-dioxin (TCDD), di-(2-ethylhexyl) phthalate (DEHP) and diethylstilbestrol (DES) have been reported to impair spermatogenesis in animal studies due to increased apoptosis (Nonclercq et al., 1996; Schultz et al., 2003; Alam et al., 2010; Erkekoglu et al., 2012). The decrease in spermatocytes at the 10,000 ppm dose in this study may be due to the observed increased apoptosis at that dose. The lack of an effect on normalized testes weight could argue that the apoptosis is a result of the more generalized toxicity of the compound.

Increased apoptosis may be caused by metabolites of HMB. Studies have suggested that metabolites of HMB have estrogenic activity in rats and in MCF-7 human breast cell lines (Okereke et al., 1993; Nakagawa and Suzuki, 2002; Suzuki et al., 2005). DHB, DHMB, and THB are generated from HMB, and their order of estrogenic potency is generally DHB > THB > DHMB (Okereke et al., 1993; Nakagawa and Suzuki, 2002; Suzuki et al., 2005). The present study detected only HMB and DHB in the serum of dams (Table 3). Some studies have suggested that the metabolites, especially DHB, may be more potent estrogens than the parent compound. In addition, Okereke et al. (1993) showed that, following treatment with gavage HMB, the testes have the second largest amount of total DHB of seven tissues tested. Thus, it is possible that DHB is responsible for impaired spermatogenesis in rat male offspring. Subchronic oral administration of DHB has been shown to decrease postnatal growth and reduce liver and kidney weights in rats (Christian, 1983) and to inhibit testosterone production from substrates in ex vivo studies using rat or mouse testes (Nashev et al., 2010). However, no detailed studies in animals regarding the effects of DHB on male reproduction are currently available. Further studies are necessary to determine the effect of intermediates of HMB metabolism on the male reproductive system.

Testosterone levels in male offspring were decreased at 3,000 ppm dose (Fig. 7). Since testosterone production is very low and Leydig cells are not mature (Picut et al., 2014), it is hard to determine the effect of maternal and lactational exposure to HMB on testosterone production. However, DHB, one of the metabolites of HMB, may influence to testosterone production (Nashev et al., 2010). Additional experiments with mature rats are needed to confirm this.

Thyroid hormones are important for growth and differentiation of many organs (Oppenheimer et al., 1987; Kress et al., 2009). Thyroid stimulating hormone (TSH) and T3 levels in PND 23 male rat offspring were measured to clarify if the effects of maternal and lactational exposure to HMB on offspring body weights were due to a reduction of thyroid hormone levels. TSH levels were slightly, but not statistically, increased in rats treated with HMB (Supplemental Fig. 1A). T3 levels were reduced by 38% and 44% in the 25,000 and 50,000 ppm HMB dose groups, respectively; however, these differences were not statistically significant probably due to the small sample number (Supplemental Fig. 1B). Results such as increased TSH and decreased T3 levels in rats treated with HMB may be caused by the feedback system in the thyroid which reduces T3 levels (Hood et al., 1999). Additional experiments with a larger sample size are needed to confirm this.

Conclusion

In conclusion, few adverse effects in rat dams and offspring dosed maternally and lactationally with HMB in chow from GD 6 to PND 23 were observed at doses of HMB of 10,000 ppm or less. A comparison of serum HMB levels in these rats to serum and urinary levels in humans suggest that exposure to HMB is probably not associated with adverse effects on the reproductive system. In the higher dose groups, it is possible that HMB produced a delay in postnatal growth which could have adversely affected reproductive organ development; however, it is not clear. Further work is needed to clarify the possible decreases in spermatogenesis and folliculogenesis observed in the current study.

Supplementary Material

Supp FigureS1. Supplemental Fig. 1 Serum thyroid-stimulating hormone (TSH) (A) and T3 (B) in PND 23 male rats.

(A) Values are expressed as mean ± SEM of 3-4 litters per group. (B) Values are expressed as mean ± SEM of 4-5 litters per group.

Supp TableS1-S3

Acknowledgments

The authors thank Ms. Anna J. Williams and Ms. Candee Teitel for helping with sample collection. The authors also thank Ms. Florene J. Lewis, Ms. Felita F. Reynolds, and the NCTR animal care technicians for excellent animal care. The author (N.N.) thanks Dr. Mariko Shirota for useful comments on the female study, Ms. Lisa D. Freeman for helping in the preparation of paraffin sections, and Ms. Sheila M. Peters and Ms. Lynne D. Finister at the FDA Biosciences Library, NCTR Branch, for helping in manuscript preparation. Also, the author (N.N.) dedicates this manuscript to the late Mr. Gene Alvin White as a token of gratitude.

Disclaimer: The views expressed are those of the authors and do not represent the views of the Food and Drug Administration.

Disclosure of funding

This study was funded from NCTR (E0745502); tissues were collected from a study funded by an Interagency Agreement between FDA/NCTR and the NIEHS/NTP (FDA 224-12-0003; NIH AES12013).

Abbreviations

HMB

2-hydroxy-4-methoxybenzophenone

DHB

2,4-dihydroxybenzophenone

THB

2,3,4-trihydroxybenzophenone

DHMB

2,2-dihydroxy-4-methoxybenzophenone

BP-2

benzophenone-2

Footnotes

Conflict of interest

The authors declare that there are no conflicts of interest.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supp FigureS1. Supplemental Fig. 1 Serum thyroid-stimulating hormone (TSH) (A) and T3 (B) in PND 23 male rats.

(A) Values are expressed as mean ± SEM of 3-4 litters per group. (B) Values are expressed as mean ± SEM of 4-5 litters per group.

NIHMS661100-supplement-Supp_FigureS1.pdf (12.7KB, pdf)
Supp TableS1-S3
NIHMS661100-supplement-Supp_TableS1-S3.doc (75KB, doc)

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