Robert L. Sprando

Methods for assessing sperm motility, morphology, and counts in the rat, rabbit, and dog: A consensus report☆

Reproductive toxicity studies are increasingly including assessments of sperm parameters including motility, morphology, and counts. While these assessments can provide valuable information for the determination of potential reproductive toxicity, the methods for conducting the assessments have not been well developed in all laboratories and are continually evolving. The use of different methods in different laboratories makes comparison of data among laboratories difficult. To address the differences in methods, a working group was convened to discuss methods currently in use, share data, and try to reach consensus about optimal methods for assessing sperm parameters in rats, rabbits, and dogs. This article presents the consensus report, as well as future research needs, with the hope that optimized common methods will aid in the detection of reproductive effects and enhance interlaboratory comparisons.

Application of nanotechnology in cosmetics.

The National Nanotechnology Initiative (NNI), established in 2001 to coordinate Federal nanotechnology research and development, defines nanotechnology as i) research and technology development at the atomic, molecular, or macromolecular levels in the length scale of approximately the 1to 100-nanometer range; ii) creating and using structures, devices, and systems that have novel properties and functions because of their small and/or intermediate size; and iii) the ability to control or manipulate on the atomic scale (1). Generally speaking, nanotechnology can be defined as the application of scientific and engineering principles to make and utilize very small things. Nanotechnology is moving into the centre of world-wide public attention because of its broad range of applications which could dramatically impact both the scientific community and the commercial marketplace. Nanotechnology applications have been applied across biomedical, optical, electronic, mechanical, and chemical fields as well as in consumer goods such as foods and cosmetics. Nanotechnology is merging with information technology, biology and social sciences and is expected to reinvigorate discoveries and innovations in many areas of the economy. Nanomedicine is dominated by nanoparticulate drug delivery systems because of their ability to cross biological barriers, accumulate at tumor sites and/or increase the solubility of drugs. Approved nanotechnology-based products include nanoscale liposomal doxorubicin (Doxil®/ Myocet TM, Ortho Biotech, Bridgewater, NJ, USA/Zeneus Pharma, Oxford, UK) for breast cancer and kaposi sacoma, lipid micelles of estradiol (Estrasorb ®, Novavax, Malvern, PA, USA) for menopausal therapy, and nanocrystalline sirolimus (Rapamune, Elan Drug Delivery, Wyeth Pharmaceuticals, Collegeville, PA, USA) for immunosuppression.

Vibrio cholerae hemolysin is required for lethality, developmental delay, and intestinal vacuolation in Caenorhabditis elegans.

Background Cholera toxin (CT) and toxin-co-regulated pili (TCP) are the major virulence factors of Vibrio cholerae O1 and O139 strains that contribute to the pathogenesis of disease during devastating cholera pandemics. However, CT and TCP negative V. cholerae strains are still able to cause severe diarrheal disease in humans through mechanisms that are not well understood. Methodology/Principal Findings To determine the role of other virulence factors in V. cholerae pathogenesis, we used a CT and TCP independent infection model in the nematode Caenorhabditis elegans and identified the hemolysin A (hlyA) gene as a factor responsible for animal death and developmental delay. We demonstrated a correlation between the severity of infection in the nematode and the level of hemolytic activity in the V. cholerae biotypes. At the cellular level, V. cholerae infection induces formation of vacuoles in the intestinal cells in a hlyA dependent manner, consistent with the previous in vitro observations. Conclusions/Significance Our data strongly suggest that HlyA is a virulence factor in C. elegans infection leading to lethality and developmental delay presumably through intestinal cytopathic changes.

Testing the potential of sodium fluoride to affect spermatogenesis : a morphometric study

This study provides quantitative information on the effect of sodium fluoride (NaF) on the testes of F1 generation male rats exposed in utero and during lactation to NaF at one of four concentrations (25, 100, 175, 250 ppm). At weaning, the F1 generation males were exposed to NaF in their drinking water for 14 weeks, after which time testicular tissues were perfusion-fixed with glutaraldehyde and observed after being embedded in plastic. The seminiferous tubules comprised 89%, 87%, 88%, 88% and 88% of the total testis volume while the interstitial space occupied 9.3%, 11.2%, 10.2%, 9.8% and 9.9% of the total testis volume for the 0, 25, 100, 175 and 250 ppm NaF treatment groups, respectively. Statistically significant differences between control and NaF-treated rats were not observed with respect to absolute volume of the seminiferous tubules, interstitial space, Leydig cells, blood vessels boundary layer, lymphatic space, macrophages, tubular lumen or absolute tubular length and absolute tubular surface area, mean Sertoli cell nucleoli number per tubular cross-section, mean seminiferous tubule diameter and the mean height of the seminiferous epithelium. A statistically significant decrease in the absolute volume and volume percent of the lymphatic endothelium was observed in the 175 and 250 ppm NaF-treated groups and in the testicular capsule in the 100 ppm NaF-treated groups. The significance of this finding is unknown at the present time. Overall, the quantitative information obtained suggests that exposure to NaF at the doses used in the present study does not adversely affect testis structure or spermatogenesis in the rat.

Comparative genotoxicity of nanosilver in human liver HepG2 and colon Caco2 cells evaluated by fluorescent microscopy of cytochalasin B-blocked micronucleus formation

As a consequence of the increased use of silver nanoparticles in food, food contact materials, dietary supplements and cosmetics to prevent fungal and bacterial growth, there is a need for validated rapid screening methods to assess the safety of nanoparticle exposure. This study evaluated two widely used in vitro cell culture models, human liver HepG2 cells and human colon Caco2 cells, as tools for assessing the potential genotoxicity of 20‐nm nanosilver. The average silver nanoparticle size as determined by transmission electron microscopy (TEM) was 20.4 nm. Dynamic light scattering (DLS) analysis showed no large agglomeration of the silver nanoparticles. The silver concentration in a 20‐nm nanosilver solution determined by the inductively coupled plasma–mass spectrometry (ICP‐MS) analysis was 0.962 mg ml−1. Analysis by ICP‐MS and TEM demonstrated the uptake of 20‐nm silver by both HepG2 and Caco2 cells. Genotoxicity was determined by the cytochalasin B‐blocked micronucleus assay with acridine orange staining and fluorescence microscopy. Concentration‐ and time‐dependent increases in the frequency of binucleated cells with micronuclei induced by the nanosilver was observed in the concentration range of 0.5 to 15 µg ml−1 in both HepG2 and Caco2 cells compared with the control. Our results indicated that HepG2 cells were more sensitive than Caco2 cells in terms of micronuclei formation induced by nanosilver exposure. In summary, the results of this study indicate that the widely used in vitro models, HepG2 and Caco2 cells in culture, represent potential screening models for prediction of genotoxicity of silver nanoparticles by in vitro micronucleus assay. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

Testing the potential of flaxseed to affect spermatogenesis: morphometry.

Quantitative information was collected on male reproductive effects of maternal and postnatal dietary exposure to flaxseed (20 or 40%), flaxmeal (13 or 26%) or standard NIH AIN-93 feed (0% flaxseed control). Measurements were made on the testes of F1 generation males rats (1) whose mothers were exposed to the diets designated above, and (2) who, after weaning, were placed on the same diet as their mothers for an additional 70 days. The seminiferous tubules comprised 86%, 84%, 84%, 84% and 85% of the total testis volume while the interstitial space comprised 12%, 14%, 14%, 14%, 13% of the total testis volume for the 0% flaxseed/flaxmeal, 20% flaxseed, 13% flaxmeal, 40% flaxseed and 26% flaxmeal groups, respectively. Statistically significant decreases in the absolute volume of the seminiferous tubules were observed in the 20% and 40% flaxseed-treated groups when these groups were compared to controls. Borderline statistically significant differences were also observed when Sertoli cell nucleolar number per tubular cross-section were compared in the 13% flaxmeal and 20% flaxseed treatment groups. These effects were not considered biologically significant because other parameters of male reproductive function appeared normal. Overall, the quantitative information obtained suggests that exposure to flaxseed/flaxmeal at the doses used in the present study does not adversely affect testis structure or spermatogenesis in the rat.

Genomic Analysis of Stress Response Against Arsenic in Caenorhabditis elegans

Arsenic, a known human carcinogen, is widely distributed around the world and found in particularly high concentrations in certain regions including Southwestern US, Eastern Europe, India, China, Taiwan and Mexico. Chronic arsenic poisoning affects millions of people worldwide and is associated with increased risk of many diseases including arthrosclerosis, diabetes and cancer. In this study, we explored genome level global responses to high and low levels of arsenic exposure in Caenorhabditis elegans using Affymetrix expression microarrays. This experimental design allows us to do microarray analysis of dose-response relationships of global gene expression patterns. High dose (0.03%) exposure caused stronger global gene expression changes in comparison with low dose (0.003%) exposure, suggesting a positive dose-response correlation. Biological processes such as oxidative stress, and iron metabolism, which were previously reported to be involved in arsenic toxicity studies using cultured cells, experimental animals, and humans, were found to be affected in C. elegans. We performed genome-wide gene expression comparisons between our microarray data and publicly available C. elegans microarray datasets of cadmium, and sediment exposure samples of German rivers Rhine and Elbe. Bioinformatics analysis of arsenic-responsive regulatory networks were done using FastMEDUSA program. FastMEDUSA analysis identified cancer-related genes, particularly genes associated with leukemia, such as dnj-11, which encodes a protein orthologous to the mammalian ZRF1/MIDA1/MPP11/DNAJC2 family of ribosome-associated molecular chaperones. We analyzed the protective functions of several of the identified genes using RNAi. Our study indicates that C. elegans could be a substitute model to study the mechanism of metal toxicity using high-throughput expression data and bioinformatics tools such as FastMEDUSA.

The Effect of Vomitoxin (Deoxnivalenol) on Testicular Morphology, Testicular Spermatid Counts and Epididymal Sperm Counts in IL-6KO [B6129-IL6 〈tmlKopf〉 (IL-6 gene deficient)] and WT [B6129F2 (wild type to B6129-IL6 with an intact IL-6 gene)] mice

The potential of vomitoxin (VT) to affect testicular morphology and testicular and epididymal sperm counts was assessed in three strains of mice: IL-6KO [B6129-IL6 (tmlKopf) (IL-6 gene deficient)], WT [B6129F2 (wild type to B6129-IL6 with an intact IL-6 gene)] and B6C3F1 mice in a 90-day feeding study. The treated mice received VT at a concentration of 10 ppm in their diet. The body weight of VT-treated animals was significantly reduced compared with control animals. Slight changes, not statistically significant, were observed in relative testis weight and testicular spermatid counts. Histological changes were not apparent in the testes of VT-treated animals. The diameter of the seminiferous tubules, the height of the seminiferous epithelium and the number of Sertoli cell nucleoli per cross-sectioned seminiferous tubule in the VT-treated groups were not significantly different from their respective untreated controls. The IL-6KO and B6C3F1 VT-treated mice had significantly reduced cauda epididymal weights compared with their respective controls. These changes were not attributed to decreased sperm counts and this finding suggests that VT may exert an adverse affect on the epididymis.

Human kidney proximal tubule cells are vulnerable to the effects of Rauwolfia serpentina

Rauwolfia serpentina (or Snake root plant) is a botanical dietary supplement marketed in the USA for maintaining blood pressure. Very few studies have addressed the safety of this herb, despite its wide availability to consumers. Its reported pleiotropic effects underscore the necessity for evaluating its safety. We used a human kidney cell line to investigate the possible negative effects of R. serpentina on the renal system in vitro, with a specific focus on the renal proximal tubules. We evaluated cellular and mitochondrial toxicity, along with a variety of other kidney-specific toxicology biomarkers. We found that R. serpentina was capable of producing highly detrimental effects in our in vitro renal cell system. These results suggest more studies are needed to investigate the safety of this dietary supplement in both kidney and other target organ systems.

Evaluation of “Dream Herb,” Calea zacatechichi, for Nephrotoxicity Using Human Kidney Proximal Tubule Cells

A recent surge in the use of dietary supplements, including herbal remedies, necessitates investigations into their safety profiles. “Dream herb,” Calea zacatechichi, has long been used in traditional folk medicine for a variety of purposes and is currently being marketed in the US for medicinal purposes, including diabetes treatment. Despite the inherent vulnerability of the renal system to xenobiotic toxicity, there is a lack of safety studies on the nephrotoxic potential of this herb. Additionally, the high frequency of diabetes-associated kidney disease makes safety screening of C. zacatechichi for safety especially important. We exposed human proximal tubule HK-2 cells to increasing doses of this herb alongside known toxicant and protectant control compounds to examine potential toxicity effects of C. zacatechichi relative to control compounds. We evaluated both cellular and mitochondrial functional changes related to toxicity of this dietary supplement and found that even at low doses evidence of cellular toxicity was significant. Moreover, these findings correlated with significantly elevated levels of nephrotoxicity biomarkers, lending further support for the need to further scrutinize the safety of this herbal dietary supplement.