Harnessing genomics to identify environmental determinants of heritable disease.

Next-generation sequencing technologies can now be used to directly measure heritable de novo DNA sequence mutations in humans. However, these techniques have not been used to examine environmental factors that induce such mutations and their associated diseases. To address this issue, a working group on environmentally induced germline mutation analysis (ENIGMA) met in October 2011 to propose the necessary foundational studies, which include sequencing of parent-offspring trios from highly exposed human populations, and controlled dose-response experiments in animals. These studies will establish background levels of variability in germline mutation rates and identify environmental agents that influence these rates and heritable disease. Guidance for the types of exposures to examine come from rodent studies that have identified agents such as cancer chemotherapeutic drugs, ionizing radiation, cigarette smoke, and air pollution as germ-cell mutagens. Research is urgently needed to establish the health consequences of parental exposures on subsequent generations.

Indoor Air Pollution and the Health of Vulnerable Groups: A Systematic Review Focused on Particulate Matter (PM), Volatile Organic Compounds (VOCs) and Their Effects on Children and People with Pre-Existing Lung Disease.

Air pollution affects health, but much of the focus to this point has been on outdoor air. Higher indoor pollution is anticipated due to increasingly energy-efficient and less leaky buildings together with more indoor activities. Studies of indoor air pollution focusing on children and people with respiratory disease from the database Web of Science (1991-2021) were systemically reviewed according to the PRISMA guidelines, with 69 studies included in the final selection. Emissions from building materials affected indoor air quality, and ventilation also had an influence. The main indoor air pollutants are Volatile Organic Compounds (VOCs) and Particulate Matter (PM). PM sources included smoking, cooking, heating, candles, and insecticides, whereas sources of coarse particles were pets, housework and human movements. VOC sources included household products, cleaning agents, glue, personal care products, building materials and vehicle emissions. Formaldehyde levels were particularly high in new houses. Personal exposure related to both indoor and outdoor pollutant levels, highlighting home characteristics and air exchange rates as important factors. Temperature, humidity, educational level, air purifiers and time near sources were also related to personal exposure. There was an association between PM and Fractional exhaled Nitric Oxide (FeNO), lung function, oxygen saturation, childhood asthma and symptoms of chronic obstructive pulmonary disease (COPD) patients. High VOCs were associated with upper airways and asthma symptoms and cancer. Effective interventional studies for PM in the future might focus on human behavior together with air purifiers and increased ventilation, whereas VOC interventions might center more on building materials and household products, alongside purification and ventilation.

Nanotechnology in Immunotherapy for Type 1 Diabetes: Promising Innovations and Future Advances.

Diabetes is a chronic condition which affects the glucose metabolism in the body. In lieu of any clinical "cure," the condition is managed through the administration of pharmacological aids, insulin supplements, diet restrictions, exercise, and the like. The conventional clinical prescriptions are limited by their life-long dependency and diminished potency, which in turn hinder the patient's recovery. This necessitated an alteration in approach and has instigated several investigations into other strategies. As Type 1 diabetes (T1D) is known to be an autoimmune disorder, targeting the immune system in activation and/or suppression has shown promise in reducing beta cell loss and improving insulin levels in response to hyperglycemia. Another strategy currently being explored is the use of nanoparticles in the delivery of immunomodulators, insulin, or engineered vaccines to endogenous immune cells. Nanoparticle-assisted targeting of immune cells holds substantial potential for enhanced patient care within T1D clinical settings. Herein, we summarize the knowledge of etiology, clinical scenarios, and the current state of nanoparticle-based immunotherapeutic approaches for Type 1 diabetes. We also discuss the feasibility of translating this approach to clinical practice.

A Concise Review: The Role of Stem Cells in Cancer Progression and Therapy.

The properties of cancer stem cells (CSCs) have recently gained attention as an avenue of intervention for cancer therapy. In this review, we highlight some of the key roles of CSCs in altering the cellular microenvironment in favor of cancer progression. We also report on various studies in this field which focus on transformative properties of CSCs and their influence on surrounding cells or targets through the release of cellular cargo in the form of extracellular vesicles. The findings from these studies encourage the development of novel interventional therapies that can target and prevent cancer through efficient, more effective methods. These methods include targeting immunosuppressive proteins and biomarkers, promoting immunization against tumors, exosome-mediated CSC conversion, and a focus on the quiescent properties of CSCs and their role in cancer progression. The resulting therapeutic benefit and transformative potential of these novel approaches to stem cell-based cancer therapy provide a new direction in cancer treatment, which can focus on nanoscale, molecular properties of the cellular microenvironment and establish a more precision medicine-oriented paradigm of treatment.

3D in vivo Magnetic Particle Imaging of Human Stem Cell-Derived Islet Organoid Transplantation Using a Machine Learning Algorithm.

Stem cell-derived islet organoids constitute a promising treatment of type 1 diabetes. A major hurdle in the field is the lack of appropriate in vivo method to determine graft outcome. Here, we investigate the feasibility of in vivo tracking of transplanted stem cell-derived islet organoids using magnetic particle imaging (MPI) in a mouse model. Human induced pluripotent stem cells-L1 were differentiated to islet organoids and labeled with superparamagnetic iron oxide nanoparticles. The phantoms comprising of different numbers of labeled islet organoids were imaged using an MPI system. Labeled islet organoids were transplanted into NOD/scid mice under the left kidney capsule and were then scanned using 3D MPI at 1, 7, and 28 days post transplantation. Quantitative assessment of the islet organoids was performed using the K-means++ algorithm analysis of 3D MPI. The left kidney was collected and processed for immunofluorescence staining of C-peptide and dextran. Islet organoids expressed islet cell markers including insulin and glucagon. Image analysis of labeled islet organoids phantoms revealed a direct linear correlation between the iron content and the number of islet organoids. The K-means++ algorithm showed that during the course of the study the signal from labeled islet organoids under the left kidney capsule decreased. Immunofluorescence staining of the kidney sections showed the presence of islet organoid grafts as confirmed by double staining for dextran and C-peptide. This study demonstrates that MPI with machine learning algorithm analysis can monitor islet organoids grafts labeled with super-paramagnetic iron oxide nanoparticles and provide quantitative information of their presence in vivo.

Artificial Intelligence Analysis of Magnetic Particle Imaging for Islet Transplantation in a Mouse Model.

PURPOSE: Current approaches to quantification of magnetic particle imaging (MPI) for cell-based therapy are thwarted by the lack of reliable, standardized methods of segmenting the signal from background in images. This calls for the development of artificial intelligence (AI) systems for MPI analysis. PROCEDURES: We utilize a canonical algorithm in the domain of unsupervised machine learning, known as K-means++, to segment the regions of interest (ROI) of images and perform iron quantification analysis using a standard curve model. We generated in vitro, in vivo, and ex vivo data using islets and mouse models and applied the AI algorithm to gain insight into segmentation and iron prediction on these MPI data. In vitro models included imaging the VivoTrax-labeled islets in varying numbers. In vivo mouse models were generated through transplantation of increasing numbers of the labeled islets under the kidney capsule of mice. Ex vivo data were obtained from the MPI images of excised kidney grafts. RESULTS: The K-means++ algorithms segmented the ROI of in vitro phantoms with minimal noise. A linear correlation between the islet numbers and the increasing prediction of total iron value (TIV) in the islets was observed. Segmentation results of the ROI of the in vivo MPI scans showed that with increasing number of transplanted islets, the signal intensity increased with linear trend. Upon segmenting the ROI of ex vivo data, a linear trend was observed in which increasing intensity of the ROI yielded increasing TIV of the islets. Through statistical evaluation of the algorithm performance via intraclass correlation coefficient validation, we observed excellent performance of K-means++-based model on segmentation and quantification analysis of MPI data. CONCLUSIONS: We have demonstrated the ability of the K-means++-based model to provide a standardized method of segmentation and quantification of MPI scans in an islet transplantation mouse model.

miR-216a-targeting theranostic nanoparticles promote proliferation of insulin-secreting cells in type 1 diabetes animal model.

Aberrant expression of miRNAs in pancreatic islets is closely related to the development of type 1 diabetes (T1D). The aim of this study was to identify key miRNAs dysregulated in pancreatic islets during T1D progression and to develop a theranostic approach to modify their expression using an MRI-based nanodrug consisting of iron oxide nanoparticles conjugated to miRNA-targeting oligonucleotides in a mouse model of T1D. Isolated pancreatic islets were derived from NOD mice of three distinct age groups (3, 8 and 18-week-old). Total RNA collected from cultured islets was purified and global miRNA profiling was performed with 3D-Gene global miRNA microarray mouse chips encompassing all mouse miRNAs available on the Sanger miRBase V16. Of the miRNAs that were found to be differentially expressed across three age groups, we identified one candidate (miR-216a) implicated in beta cell proliferation for subsequent validation by RT-PCR. Alterations in miR-216a expression within pancreatic beta cells were also examined using in situ hybridization on the frozen pancreatic sections. For in vitro studies, miR-216a mimics/inhibitors were conjugated to iron oxide nanoparticles and incubated with beta cell line, ßTC-6. Cell proliferation marker Ki67 was evaluated. Expression of the phosphatase and tensin homolog (PTEN), which is one of the direct targets of miR-216a, was analyzed using western blot. For in vivo study, the miR-216a mimics/inhibitors conjugated to the nanoparticles were injected into 12-week-old female diabetic Balb/c mice via pancreatic duct. The delivery of the nanodrug was monitored by in vivo MRI. Blood glucose of the treated mice was monitored post injection. Ex vivo histological analysis of the pancreatic sections included staining for insulin, PTEN and Ki67. miRNA microarray demonstrated that the expression of miR-216a in the islets from NOD mice significantly changed during T1D progression. In vitro studies showed that treatment with a miR-216a inhibitor nanodrug suppressed proliferation of beta cells and increased the expression of PTEN, a miR-216a target. In contrast, introduction of a mimic nanodrug decreased PTEN expression and increased beta cell proliferation. Animals treated in vivo with a mimic nanodrug had higher insulin-producing functionality compared to controls. These observations were in line with downregulation of PTEN and increase in beta cell proliferation in that group. Our studies demonstrated that miR-216a could serve as a potential therapeutic target for the treatment of diabetes. miR-216a-targeting theranostic nanodrugs served as exploratory tools to define functionality of this miRNA in conjunction with in vivo MR imaging.

Elevated frequencies of micronucleated erythrocytes in infants exposed to zidovudine in utero and postpartum to prevent mother-to-child transmission of HIV.

Zidovudine-based antiretroviral therapies (ARTs) for treatment of HIV-infected pregnant women have markedly reduced mother-to-child transmission of the human immunodeficiency virus (HIV-1) from approximately 25% to <1%. However, zidovudine (ZDV; AZT), a nucleoside analogue, induces chromosomal damage, gene mutations, and cancer in animals following direct or transplacental exposure. To determine if chromosomal damage is induced by ZDV in infants exposed transplacentally, we evaluated micronucleated reticulocyte frequencies (%MN-RET) in 16 HIV-infected ART-treated mother-infant pairs. Thirteen women received prenatal ART containing ZDV; three received ART without ZDV. All infants received ZDV for 6 weeks postpartum. Venous blood was obtained from women at delivery and from infants at 1-3 days, 4-6 weeks, and 4-6 months of life; cord blood was collected immediately after delivery. Ten cord blood samples (controls) were obtained from infants of HIV-uninfected women who did not receive ART. %MN-RET was measured using a single laser 3-color flow cytometric system. Tenfold increases in %MN-RET were seen in women and infants who received ZDV-containing ART prenatally; no increases were detected in three women and infants who received prenatal ART without ZDV. Specifically, mean %MN-RET in cord blood of ZDV-exposed infants was 1.67 +/- 0.34 compared with 0.16 +/- 0.06 in non-ZDV ART-exposed infants (P = 0.006) and 0.12 +/- 0.02 in control cord bloods (P < 0.0001). %MN-RET in ZDV-exposed newborns decreased over the first 6 months of life to levels comparable to cord blood controls. These results demonstrate that transplacentalZDV exposure is genotoxic in humans. Long-term monitoring of HIV-uninfected ZDV-exposed infants is recommended to ensure their continued health.

Assessing human germ-cell mutagenesis in the Postgenome Era: a celebration of the legacy of William Lawson (Bill) Russell.

Birth defects, de novo genetic diseases, and chromosomal abnormality syndromes occur in approximately 5% of all live births, and affected children suffer from a broad range of lifelong health consequences. Despite the social and medical impact of these defects, and the 8 decades of research in animal systems that have identified numerous germ-cell mutagens, no human germ-cell mutagen has been confirmed to date. There is now a growing consensus that the inability to detect human germ-cell mutagens is due to technological limitations in the detection of random mutations rather than biological differences between animal and human susceptibility. A multidisciplinary workshop responding to this challenge convened at The Jackson Laboratory in Bar Harbor, Maine. The purpose of the workshop was to assess the applicability of an emerging repertoire of genomic technologies to studies of human germ-cell mutagenesis. Workshop participants recommended large-scale human germ-cell mutation studies be conducted using samples from donors with high-dose exposures, such as cancer survivors. Within this high-risk cohort, parents and children could be evaluated for heritable changes in (a) DNA sequence and chromosomal structure, (b) repeat sequences and minisatellites, and (c) global gene expression profiles and pathways. Participants also advocated the establishment of a bio-bank of human tissue samples from donors with well-characterized exposure, including medical and reproductive histories. This mutational resource could support large-scale, multiple-endpoint studies. Additional studies could involve the examination of transgenerational effects associated with changes in imprinting and methylation patterns, nucleotide repeats, and mitochondrial DNA mutations. The further development of animal models and the integration of these with human studies are necessary to provide molecular insights into the mechanisms of germ-cell mutations and to identify prevention strategies. Furthermore, scientific specialty groups should be convened to review and prioritize the evidence for germ-cell mutagenicity from common environmental, occupational, medical, and lifestyle exposures. Workshop attendees agreed on the need for a full-scale assault to address key fundamental questions in human germ-cell environmental mutagenesis. These include, but are not limited to, the following: Do human germ-cell mutagens exist? What are the risks to future generations? Are some parents at higher risk than others for acquiring and transmitting germ-cell mutations? Obtaining answers to these, and other critical questions, will require strong support from relevant funding agencies, in addition to the engagement of scientists outside the fields of genomics and germ-cell mutagenesis.

Mitochondrial toxicity in hearts of CD-1 mice following perinatal exposure to AZT, 3TC, or AZT/3TC in combination.

Antiretroviral therapies based on nucleoside reverse transcriptase inhibitors (NRTIs), like zidovudine (3'-azido-3'-deoxythymidine; AZT) and lamivudine ((-)2',3'-dideoxy-3'-thiacytidine; 3TC), markedly reduce mother-to-child transmission of the human immunodeficiency virus (HIV). However, AZT induces damage in nuclear DNA of mice exposed in utero and postnatally, and mitochondrial DNA (mtDNA) damage has been observed in both human and mouse neonates following perinatal exposure to AZT and AZT/3TC in combination. To provide animal data modeling the NRTI-induced heart damage reported in human infants, we treated pregnant CD-1 mice throughout gestation and treated their pups by direct gavage from postnatal day (PND) 4 through PND 28 with daily doses of 150 mg/kg body weight (bw)/day AZT, 75 mg/kg bw/day 3TC, 125/62.5 mg/kg bw/day AZT/3TC, or the vehicle control. Half the pups were euthanized on PND 28; the remainder received no further dosing, and were euthanized at week 10. Heart tissue was collected, total DNA was extracted, and mtDNA copy number relative to nuclear DNA copy number, mtDNA damage, and mtDNA mutation assays were performed using PCR-based methods. Analyses revealed increases in mtDNA lesions in 4-week-old males and females treated with AZT or 3TC, but not in 10-week-old mice, suggesting that the damage resolved after treatment ceased. Interestingly, 10-week-old females treated with AZT/3TC had significant increases in mtDNA damage. Point mutations were elevated in 10-week-old females treated with AZT or AZT/3TC, but not 3TC; no increases in mutations were seen in either gender at 4 weeks of age. Our data suggest that AZT/3TC combination treatment produces greater mtDNA damage than either agent individually, and that female mice are more sensitive than males to AZT/3TC-induced mtDNA damage.

NOVP chemotherapy for Hodgkin's disease transiently induces sperm aneuploidies associated with the major clinical aneuploidy syndromes involving chromosomes X, Y, 18, and 21.

The objective of this research was to determine whether Novantrone, Oncovin, Velban, and Prednisone (NOVP) combination chemotherapy for Hodgkin's disease increases the frequencies of the specific types of aneuploid sperm that might elevate the risk of fathering a child with one of the major clinical aneuploidy syndromes, i.e., Down (disomy 21 sperm), Edward (disomy 18 sperm), Turner (nullisomy sex sperm), XYY (disomy Y sperm), triple X (disomy X sperm), or Klinefelter (XY sperm). A four-chromosome multicolor sperm fluorescence in-situ hybridization assay that simultaneously evaluates chromosomes 18, 21, X, and Y was applied to semen provided by four healthy men and to repeated samples of eight Hodgkin's disease patients before treatment, 35-50 days after treatment to examine the effects of treatment on male meiotic cells, and 1-2 years after treatment to measure the persistence of damage. There were chromosome-specific variations in baseline frequencies and significant inductions of all of the detectable types of sperm aneuploidies: XY sperm (14-fold increase), disomy 18 (7-fold), nullisomy sex (3-fold), disomy 21 (3-fold), and disomy X and Y (approximately 2-fold each). Disomy 21 was about twice as frequent as disomy 18, and neither showed a preferential segregation with a sex chromosome. Extrapolating across the genome, approximately 18% of sperm carried a numerical abnormality after NOVP treatment of meiotic cells. Induced effects did not persist to 1-2 years after treatment, suggesting that persistent spermatogonial stem cells were not sensitive to NOVP. These findings establish the hypothesis that conception shortly after certain chemotherapies can transiently increase the risks of fathering aneuploid pregnancies that terminate during development or result in the birth of children with major human aneuploidy syndromes.

Meiotic interstrand DNA damage escapes paternal repair and causes chromosomal aberrations in the zygote by maternal misrepair.

De novo point mutations and chromosomal structural aberrations (CSA) detected in offspring of unaffected parents show a preferential paternal origin with higher risk for older fathers. Studies in rodents suggest that heritable mutations transmitted from the father can arise from either paternal or maternal misrepair of damaged paternal DNA, and that the entire spermatogenic cycle can be at risk after mutagenic exposure. Understanding the susceptibility and mechanisms of transmission of paternal mutations is important in family planning after chemotherapy and donor selection for assisted reproduction. We report that treatment of male mice with melphalan (MLP), a bifunctional alkylating agent widely used in chemotherapy, induces DNA lesions during male mouse meiosis that persist unrepaired as germ cells progress through DNA repair-competent phases of spermatogenic development. After fertilization, unrepaired sperm DNA lesions are mis-repaired into CSA by the egg's DNA repair machinery producing chromosomally abnormal offspring. These findings highlight the importance of both pre- and post-fertilization DNA repair in assuring the genomic integrity of the conceptus.

Approaches for identifying germ cell mutagens: Report of the 2013 IWGT workshop on germ cell assays(☆).

This workshop reviewed the current science to inform and recommend the best evidence-based approaches on the use of germ cell genotoxicity tests. The workshop questions and key outcomes were as follows. (1) Do genotoxicity and mutagenicity assays in somatic cells predict germ cell effects? Limited data suggest that somatic cell tests detect most germ cell mutagens, but there are strong concerns that dictate caution in drawing conclusions. (2) Should germ cell tests be done, and when? If there is evidence that a chemical or its metabolite(s) will not reach target germ cells or gonadal tissue, it is not necessary to conduct germ cell tests, notwithstanding somatic outcomes. However, it was recommended that negative somatic cell mutagens with clear evidence for gonadal exposure and evidence of toxicity in germ cells could be considered for germ cell mutagenicity testing. For somatic mutagens that are known to reach the gonadal compartments and expose germ cells, the chemical could be assumed to be a germ cell mutagen without further testing. Nevertheless, germ cell mutagenicity testing would be needed for quantitative risk assessment. (3) What new assays should be implemented and how? There is an immediate need for research on the application of whole genome sequencing in heritable mutation analysis in humans and animals, and integration of germ cell assays with somatic cell genotoxicity tests. Focus should be on environmental exposures that can cause de novo mutations, particularly newly recognized types of genomic changes. Mutational events, which may occur by exposure of germ cells during embryonic development, should also be investigated. Finally, where there are indications of germ cell toxicity in repeat dose or reproductive toxicology tests, consideration should be given to leveraging those studies to inform of possible germ cell genotoxicity.

Mitochondrial damage revealed by morphometric and semiquantitative analysis of mouse pup cardiomyocytes following in utero and postnatal exposure to zidovudine and lamivudine.

Zidovudine (ZDV), an antiretroviral drug used alone or in combination with other antiretroviral agents to treat HIV-infected pregnant women and their newborn infants, effectively reduces mother-to-child transmission of the virus. That myopathy and cardiomyopathy, related to mitochondrial damage, develop in some adults chronically treated with ZDV has long been known; recently, reports have suggested that similar adverse effects may occur in some infants exposed perinatally. Using a mouse model of human neonatal exposure, we treated pregnant CD-1 mice twice daily with doses of 75 mg/kg ZDV plus 37.5 mg/kg lamivudine throughout gestation and lactation; pups were exposed by direct gavage beginning postnatal day (PND) 4 and sacrificed on PND 28. Hearts were removed rapidly, and ventricles were processed for electron microscopy. Morphometric and semiquantitative morphological analyses were performed on three micrographs from each of three blocks from each of three females and three males from the control and treated groups. Treated mice showed significant increases in the mean area and decreases in the mean number of cardiomyocytic mitochondria compared to controls. We observed clusters of damaged mitochondria more frequently in treated animals than in controls; damage included fragmentation and loss of cristae. These results, demonstrating alterations in cardiomyocytic mitochondria of mice exposed in utero and postnatally, may model cardiac damage reported in human infants similarly exposed to ZDV. Critical insights derived from animal-model data like these may be used to mitigate risks to thousands of human infants receiving essential lifesaving therapy with antiretroviral drugs.

Genetic damage detected in CD-1 mouse pups exposed perinatally to 3'-azido-3'-deoxythymidine and dideoxyinosine via maternal dosing, nursing, and direct gavage.

Human immunodeficiency virus (HIV)-infected pregnant women are administered nucleoside-analogue antiretrovirals to reduce maternal-infant viral transmission. The current protocol recommends treating newborns for 6 additional weeks postpartum. The treatment is effective, but the risk of drug-induced chromosomal damage in neonates remains undefined. We used a mouse model to investigate this concern. In a multigeneration reproductive toxicity study, female CD-1 mice received 3'-azido-3'-deoxythymidine (AZT) and dideoxyinosine (ddI) (50/250, 75/375, 150/750 mg/kg/day AZT/ddI) by gavage twice daily in equal fractions beginning prior to mating and continuing throughout gestation and lactation. Direct pup dosing (same regimen) began on postnatal day (PND) 4. Peripheral blood erythrocytes of male pups were screened for micronuclei, markers of chromosomal damage, on PNDs 1, 4, 8, and 21. Extraordinary increases in micronucleated cells were noted in pups for each treatment group at each sampling time; treated dams exhibited smaller yet significant increases in micronucleated erythrocytes. The frequencies of micronucleated cells in untreated pups were higher than in the untreated dams, and all pups had markedly elevated levels of circulating reticulocytes compared to dams. These observations suggest that fetal and neonatal mouse hematopoietic precursor cells have heightened sensitivity to genotoxic agents, perhaps due to rapid cell proliferation during the perinatal period of development. The amount of genetic damage observed in treated pups raises concern for the potential of similar damage in humans. Investigations of chromosomal integrity in exposed newborns and children are recommended.

Genetic damage detected in CD-1 mouse pups exposed perinatally to 3'-azido-3'-deoxythymidine or dideoxyinosine via maternal dosing, nursing, and direct gavage: II. Effects of the individual agents compared to combination treatment.

We previously reported extraordinary increases in micronucleated erythrocytes in CD-1 mouse pups exposed to 3'-azido-3'-deoxythymidine (AZT) and dideoxyinosine (ddI; 50/250, 75/375, 150/750 mg/kg/day AZT/ddI) by gavage throughout gestation and lactation, followed by direct pup dosing beginning postnatal day (PND) 4 (Bishop et al. [2004]: Environ Mol Mutagen 43: 3-9). That study was conducted to explore the potential for genetic damage in newborns exposed perinatally to antiretrovirals in order to reduce maternal-infant transmission of HIV-1. Because dramatic increases in frequencies of micronucleated erythrocytes were seen in exposed pups, additional studies were conducted to clarify the relative contribution of each drug to the observed damage. Pregnant CD-1 mice were administered AZT (50, 75, 150 mg/kg/day) or ddI (250, 375, 750 mg/kg/day) by gavage twice daily in equal fractions beginning prior to mating and continuing throughout gestation and lactation. Direct pup dosing (same regimens) began on PND 4. Peripheral blood erythrocytes of male pups were screened for micronuclei on PNDs 1, 4, 8, and 21. Significant increases in micronucleated erythrocytes were observed in pups and dams exposed to AZT at all doses and sampling times. The highest micronucleus levels were observed in pups on PND 8 after the initiation of direct dosing. In contrast, effects seen in pups and dams treated with ddI were minimal. These results demonstrate that AZT, a component of many anti-HIV combination therapies, induces chromosomal damage in perinatally exposed neonatal mice. Comparison of micronucleated cell frequencies induced by AZT alone or in combination with ddI suggests that ddI potentiates AZT-induced chromosomal damage following direct exposure.

Mouse bone marrow micronucleus test results do not predict the germ cell mutagenicity of N-hydroxymethylacrylamide in the mouse dominant lethal assay.

N-Hydroxymethylacrylamide (NHMA), a mouse carcinogen inactive in the Salmonella assay and mouse micronucleus (MN) assay, was tested for reproductive effects in a mouse continuous breeding study. In that study, increased embryonic deaths were observed after 13 weeks exposure of parental animals to NHMA via drinking water (highest dose, 360 ppm); the results indicated the possible induction of chromosome damage in germ cells of treated males. An additional mouse MN test was conducted using a 31-day treatment period to better match the dosing regimen used in the breeding study; the results were negative. Additional studies were conducted to explore the germ cell activity of NHMA. A male mouse dominant lethal study was conducted using a single intraperitoneal injection of 150 mg/kg NHMA; the results were negative. A follow-up study was conducted using fractionated dosing, 50 mg/kg/day for 5 days; again, no increase in dominant lethal mutations was observed. NHMA (180-720 ppm) was then administered to male mice in drinking water for 13 weeks, during which three sets of matings occurred. Two weeks after mating, females were killed and the uterine contents were analyzed. Large, dose-related increases in dominant lethal mutations were observed with increasing length of exposure. The magnitude of the increases stabilized after 8 weeks of treatment. However, the frequency of micronucleated peripheral blood erythrocytes was not elevated in mice treated for 13 weeks with NHMA in drinking water. Thus, NHMA appears to be unique in inducing genetic damage in germ cells but not somatic cells of male mice.

Female-specific reproductive toxicities following preconception exposure to xenobiotics.

Females do differ from males in their germ cell and general reproductive responses to toxicants. Chromatin structure is perhaps one factor that contributes to sex differences in germ cell response to toxicants. Differences in available targets likely contribute to response differences between sexes as well as to within sex differences between germ cell stages. It is important to consider these differences when conducting reproductive toxicity studies and interpreting the results.

Determination of the di-(2-ethylhexyl) phthalate NOAEL for reproductive development in the rat: importance of the retention of extra animals to adulthood.

Deriving No Observed Adverse Effect Level (NOAEL) or benchmark dose is important for risk assessment and can be influenced by study design considerations. In order to define the di-(2-ethylhexyl) phthalate (DEHP) dose-response curve for reproductive malformations, we retained more offspring to adulthood to improve detection of these malformations in the reproductive assessment by continuous breeding study design. Sprague-Dawley rats were given a dietary administration of 1.5 (control), 10, 30, 100, 300, 1000, 7500, and 10,000 ppm DEHP. Male pups were evaluated for gross reproductive tract malformations (RTMs) associated with the "phthalate syndrome." DEHP treatment had minimal effects on P0 males. There was a statistically significant increase in F1 and F2 total RTMs (testis, epididymides, seminal vesicle, and prostate) in the 7500-ppm dose group and F1 10,000-ppm dose group. The 10,000-ppm exposed F1 males did not produce an F2 generation. The NOAEL for F1 and F2 RTM combined data, because in utero exposures were similar, were 100 ppm (4.8 mg/kg/day), which was close to the 5% response benchmark dose lower confidence limit of 142 ppm. The utility of evaluating more pups per litter was examined by generating power curves from a Monte Carlo simulation. These curves indicate a substantial increase in detection rate when three males are evaluated per litter rather than one. A 10% effect across male pups would be detected 5% of the time if one pup per litter was evaluated, but these effects would be detected 66% of the time if three pups per litter were evaluated. Taken together, this study provides a well-defined dose response of DEHP-induced RTMs and demonstrates that retention of more adult F1 and F2 males per litter, animals that were already produced, increases the ability to detect RTMs and presumably other low-incidence phenomena.

Chromosomal mosaicism in mouse two-cell embryos after paternal exposure to acrylamide.

Chromosomal mosaicism in human preimplantation embryos is a common cause of spontaneous abortions, however, our knowledge of its etiology is limited. We used multicolor fluorescence in situ hybridization painting to investigate whether paternally transmitted chromosomal aberrations result in mosaicism in mouse two-cell embryos. Paternal exposure to acrylamide, an important industrial chemical also found in tobacco smoke and generated during the cooking process of starchy foods, produced significant increases in chromosomally defective two-cell embryos, however, the effects were transient primarily affecting the postmeiotic stages of spermatogenesis. Comparisons with our previous study of zygotes demonstrated similar frequencies of chromosomally abnormal zygotes and two-cell embryos suggesting that there was no apparent selection against numerical or structural chromosomal aberrations. However, the majority of affected two-cell embryos were mosaics showing different chromosomal abnormalities in the two blastomeric metaphases. Analyses of chromosomal aberrations in zygotes and two-cell embryos showed a tendency for loss of acentric fragments during the first mitotic division of embryogenesis, whereas both dicentrics and translocations apparently underwent proper segregation. These results suggest that embryonic development can proceed up to the end of the second cell cycle of development in the presence of abnormal paternal chromosomes and that even dicentrics can persist through cell division. The high incidence of chromosomally mosaic two-cell embryos suggests that the first mitotic division of embryogenesis is prone to missegregation errors and that paternally transmitted chromosomal abnormalities increase the risk of missegregation leading to embryonic mosaicism.