Reproductive toxicants have a threshold of adversity

This paper surveys the scientific basis for the current threshold approach for reproductive hazard and risk assessment. In some regulatory areas it was recently suggested to consider reproductive toxicants under the stringent linear extrapolation risk assessment paradigm that was developed for genotoxic carcinogens. First, the current risk assessment paradigm for genotoxic carcinogens is addressed, followed by an overview of reproductive toxicology and its threshold dose approach for hazard and risk assessment, the testing procedures for assessing the reproductive toxicity of chemicals, and the derivation of conclusions on their risk assessment and Classification, Labelling and Packaging (CLP). Relevant details of testing methodologies are discussed, such as exposure time windows, parameters determined, and the coverage of the entire reproductive cycle. In addition, the dose-response relationship is considered, illustrated with several examples. It is concluded that the current risk assessment methodology for genotoxic carcinogens is a debatable worst-case scenario and that for risk assessment of reproductive toxicants the threshold dose approach remains valid.     

Reproductive toxicology of environmental toxicants: emerging issues and concerns

Environmental toxicants comprise a number of man-made organic chemicals which may resist metabolism or their metabolites may persist in the environment and accumulate in the food chain. Some of these persistent chemicals are carried over long distances via the atmospheric transport and can have biological effects in fish, wildlife and humans. In this review the relationship between structure of these chemicals, their mode of action and their possible roles in adverse developmental and reproductive processes in humans will be discussed. The focus will be on model polychlorinated biphenyls and dioxins, organochlorines, phthalates, a constituent of cigarette smoke (benzo-a-pyrene), synthetic polymers (polybrominated diphenyl ethers and polyfluorinated compounds), and a fungicide (vinclozolin).     

Food plant toxicants and safety Risk assessment and regulation of inherent toxicants in plant foods

The ADI as a tool for risk management and regulation of food additives and pesticide residues is not readily applicable to inherent food plant toxicants: The margin between actual intake and potentially toxic levels is often small; application of the default uncertainty factors used to derive ADI values, particularly when extrapolating from animal data, would prohibit the utilisation of the food, which may have an overall beneficial health effect. Levels of inherent toxicants are difficult to control; their complete removal is not always wanted, due to their function for the plant or for human health. The health impact of the inherent toxicant is often modified by factors in the food, e.g. the bioavailability from the matrix and interaction with other inherent constituents. Risk-benefit analysis should be made for different consumption scenarios, without the use of uncertainty factors. Crucial in this approach is analysis of the toxicity of the whole foodstuff. The relationship between the whole foodstuff and the pure toxicant is expressed in the `product correction factor' (PCF). Investigations in humans are essential so that biomarkers of exposure and for effect can be used to analyse the difference between animals and humans and between the food and the pure toxicant. A grid of the variables characterising toxicity is proposed, showing their inter-relationships. A flow diagram for risk estimate is provided, using both toxicological and epidemiological studies.     

Perturbation of epigenetic status by toxicants

It is becoming increasingly apparent that toxicant-induced changes in epigenetic status, particularly DNA methylation patterns, may play a role in some mechanisms of toxicity. Here, we discuss briefly the evidence that alterations in DNA methylation accompany, and may even promote, carcinogenesis induced by non-genotoxic chemicals. We also address recent findings indicating that the availability of dietary methyl donors can modulate DNA methylation levels and precipitate adverse effects.     

Toxicity of binary mixtures of reactive toxicants

This study evaluated the toxicity of binary mixtures of reactive toxicants using the Microtox test. Greater than additive effects were quite frequently observed (18%) among chemicals with different mechanisms of toxicity, and some of them were severely synergistic. The concentration-addition model, therefore, may not be appropriate for estimating the multiple toxicity of mixtures containing reactive toxicants. The slope of a chemical's concentration-response curve was found to play an important role in determining the mode of joint actions, which could be related to the complex joint action identified earlier. The results of this study have been summarized into several relationships that can be used to estimate the potential risks for mixtures with unknown toxicity. © 1996 by John Wiley & Sons, Inc.     

Assessment of a scheme for prioritizing inorganic toxicants by using signs-and-symptoms analysis

In cases of exposure to unknown substances, analyzing environmental or biological samples for elevated levels of toxicants can be extremely complex. We assessed a method for prioritizing toxic substances according to the agreement between toxic-specific and incident-reported signs and symptoms by analyzing 25 reported case histories of exposure to inorganic toxicants. In all but one case, this analysis scheme successfully prioritized toxicants for subsequent analysis. Despite its limitations, the scheme is a substantial improvement over other available methods.     

Life adversity is associated with smoking relapse after a quit attempt

Multiple cross-sectional studies have linked adverse childhood events and adult adversities to current smoking, lifetime smoking, and former smoking. To date, however, there have been no direct observational studies assessing the influence of adversities on smoking relapse. We prospectively followed 123 participants, 86 of whom were habitual smokers, from pre-quit ad libitum smoking to four weeks post‐quit. Thirty-seven non-smokers were also tested in parallel as a comparison group. Subjects provided biological samples for confirmation of abstinence status and self-report history of adversities such as abuse, neglect, family dysfunction, incarceration, and child-parent separation. They also completed mood and smoking withdrawal symptom measures. The results indicated that within non-smokers and smokers who relapsed within the first month of a quit attempt, but not abstainers, females had significantly higher adversity scores than males. Cigarette craving, which was independent from depressive affect, increased for low adversity participants, but not those with no adversity nor high adversity. These results demonstrate that sex and relapse status interact to predict adversity and that craving for nicotine may be an important additional mediator of relapse. These results add further support to the previous cross-sectional evidence of an adversity and smoking relationship. Further studies to clarify how adversity complicates smoking cessation and impacts smoking behaviors are warranted.     

Impacts of environmental toxicants on male reproductive dysfunction

Male infertility caused by exposure to environmental toxicants such as cadmium, mercury, bisphenol A (BPA) and dioxin is a global problem, particularly in industrialized countries. Studies in the testis and other organs have illustrated the importance of environmental toxicant-induced oxidative stress in mediating disruption to cell junctions. This, in turn, is regulated by the activation of PI3K/c-Src/FAK and MAPK signaling pathways, with the involvement of polarity proteins. This leads to reproductive dysfunction such as reduced sperm count and reduced quality of semen. In this review, we discuss how these findings can improve understanding of the modes of action of environmental toxicants in testicular dysfunction. Thus, specific inhibitors and/or antagonists against signaling molecules in these pathways may be able to 'reverse' and/or 'block' the disruptive effects of toxicant-induced damage. Additional studies comparing high-level acute exposure versus low-level chronic exposure to environmental toxicants are also needed to fully elucidate the underlying molecular mechanism(s) by which these toxicants disrupt male reproductive function.     

Transport of conjugates of toxicants by blood proteins

Binding of naphthol and its glucoside and glucuronide conjugates by blood proteins was studied in vitro and in vivo. Binding was found to be primarily to the albumin fraction of human blood and the binding constants were moderate to low. Both in vivo (mice) and in vitro (human) experiments suggest that a substantial portion of naphthol and two conjugates are transported in bound form to the site of elimination.     

Differential effects of olfactory toxicants on olfactory regeneration

The aim was to study the long-term response in the olfactory mucosa of NMRI mice after exposure to the olfactory toxicants dichlobenil (a herbicide) or methimazole (an antithyroid drug). Three and six months after exposure to dichlobenil (2x or 1 x 25 mg/kg i.p.), the dorsomedial part of the olfactory region showed a respiratory metaplasia with abundant invaginations and a fibrotic lamina propria. In contrast, 3 months after exposure to a toxic dose of methimazole (2 x 50 mg/kg i.p.), the olfactory neuroepithelium and lamina propria had been restored. To study the regenerative events, we used an antibody derived against growth-associated protein 43 (GAP-43), which stains immature neurons. To study epithelial differentiation and horizontal basal cells (HBCs) we used an antibody derived against some cytokeratins. Two weeks after methimazole treatment, there was a marked increase of GAP-43-stained cells in the whole olfactory region, which correlated with the observed regeneration at that time. Two weeks after dichlobenil treatment, the damaged atypical epithelium in the olfactory region showed a distinct keratin staining of basal and columnar cells whereas GAP-43-stained cells were not found. Despite a transient increase of GAP-43-stained cells in the border zone between damaged and undamaged olfactory mucosa, an expansion of a normal neuroepithelium into the damaged olfactory region was not detected in the dichlobenil-treated mice. An intact lamina propria is suggested as a prerequisite for repopulation of the neuroepithelium after toxicant-induced injury.     

Prioritization of NTP reproductive toxicants for field studies

Population studies that evaluate human reproductive impairment are time consuming, expensive, logistically difficult, and with limited resources must be prioritized to effectively prevent the adverse health effects in humans. Interactions among health scientists, unions, and industry can serve to identify populations exposed to potential hazards and develop strategies to evaluate and apply appropriate controls. This report describes a systematic method for prioritizing chemicals that may need human reproductive health field studies. Rodent reproductive toxicants identified from the National Toxicology Program (NTP) Reproductive Assessment by Continuous Breeding (RACB) protocol were prioritized on the basis of potency of toxic effect and population at risk. This model for prioritization links NTP findings with data from the National Occupational Exposure Survey (NOES) and the Hazardous Substance Data Base (HSDB) or the High Production Volume Chemical Database (HPVC) to prioritize chemicals for their potential impact on worker populations. The chemicals with the highest priority for field study were: dibutyl phthalate, boric acid, tricresyl phosphate, and N, N-dimethylformamide.     

Reproductive effects of a pegylated curcumin

Curcumin, a polyphenol derived from the rhizome turmeric, has potential as an anticancer agent. We synthesized an amphipathic/surfactant pegylated curcumin (curcumin-PEG) designed for parenteral administration. Objectives of these investigations were to assess side-effects of a therapeutic regimen of curcumin-PEG in a preclinical model. Intraperitoneal (ip) tumor burdens were reduced in athymic female mice grafted with human SKOV-3 ovarian adenocarcinoma cells and injected intravenously (iv) with curcumin-PEG. There were no gross anatomical or histopathological effects detected in non-reproductive organs. Uteri (luminal fluid imbibition) and ovaries (decreased folliculogenesis) were affected by treatment. Curcumin-PEG ip hastened the onset of puberty in immature female mice. Live births were reduced in mature females housed with males and treated iv with curcumin-PEG; mating (vaginal plugs) was not affected. Accessory gland weights, testicular testosterone concentrations, and spermatogenesis were diminished in mature male mice following iv curcumin-PEG. Estrogenic/antiandrogenic and pregnancy-disrupting effects of a water soluble/bioavailable curcumin were demonstrated.     

The development of a protocol to assess reproductive effects of toxicants in the rat

The determination that a chemical poses a reproductive risk to man typically relies upon fertility studies using rodents. However, fertility in rodents is often difficult to disrupt and more sensitive indicators of reproductive function should be included in the risk assessment process. The present discussion compares the sensitivity of fertility to other endpoints following exposure to known reproductive toxicants. In our studies rats were dosed from weaning through puberty, gestation, and lactation. The reproductive function of the male, the female, and the offspring was assessed. The effects of methoxychlor, carbendazim (MBC), dibutyl phthalate (DBP), and lindane are discussed and compared to fertility. For each compound a ratio (SR = sensitivity ratio) of the lowest effect level (LEL) for infertility or reduced fecundity to the LEL for the most sensitive physiologic endpoint was calculated. The SR should be large when a compound produces effects over a wide range of doses, but should equal unity when the dose-response curve is very steep. For methoxychlor, which blocked implantation, pubertal landmarks and estrous cyclicity provided rapid and sensitive indicators of the subsequent reproductive failure. The SR = 8 (100/12) for methoxychlor using data from females. In contrast, DBP and MBC directed altered testicular function, and for these compounds, sperm and testicular measures provided sensitive indicators of toxicity. The SR for MBC was 2 (100/50), while DBP had a SR of 1 (500/500). In the lindane study, fertility was not reduced but most of the pups (F1) died shortly after birth. The SR for lindane is equal to 0.5 (10/20). At 20 mg/kg the treated females were larger and their estrous cycles were erratic. The data from these studies indicate that no single endpoint will consistently be the most sensitive indicator of reproductive toxicity. Studies must include a number of well validated endpoints that provide a comprehensive assessment of the entire reproductive system of the male and female.     

Lethal and sublethal effects of toxicants on bumble bee populations: a modelling approach

Ecotoxicology - Pollinator decline worldwide is well-documented; globally, chemical pesticides (especially the class of pesticides known as neonicotinoids) have been implicated in hymenopteran...     

Identification of toxicants from marine sediment using effect-directed analysis

Effect-directed analysis (EDA) has been reported to be a powerful tool for the identification of the responsible toxicants in contaminated, hazardous environmental samples. The aim of this study was to investigate whether it also is possible to use currently available EDA methodology to identify potentially relevant toxicants in samples that do not pose obvious problems. For this purpose, compounds extracted from a marine sediment sample from the west coast of Sweden were separated into distinct fractions, using two preparative chromatographic techniques. One algal bioassay using Scenedesmus vacuolatus and two bacterial bioassays using Vibrio fischeri were applied as detectors of toxicity, representing acute and chronic end points. Chronic algal toxicity was a powerful tool for discriminating between toxic and nontoxic fractions, whereas acute and chronic bacterial toxicity failed to identify toxic fractions. Eight compounds were identified as potentially relevant toxicants by chemical analysis of toxic fractions: anthracene, fluoranthene, pyrene, benzo[a]anthracene, benzo[b]fluoranthene, benzo[a]pyrene, benzo[k]fluoranthene, and indeno[1,2,3-cd]pyrene.     

Implications of research on assays to characterize thyroid toxicants

Many aspects of thyroid endocrinology are very well conserved across vertebrate taxa. These aspects include thyroid hormone chemistry, the mechanism of its synthesis, and the proteins involved in these processes. In addition, the system by which the hormone is delived from the thyroid gland to target cells, including transport and regulation within the hypothalamic-pituitary-thyroid (HPT) axis, and the proteins that regulate the different components of this delivery system appear to be highly conserved across the vertebrates. Finally, the receptors that mediate thyroid hormone action and the roles thyroid hormone plays are very similar among the vertebrates. Thus, the goal of this chapter is to provide a brief synopsis of the literature supporting existing screening and testing strategies in different vertebrate taxa, and to provide insight into the strengths, weaknesses, and likely changes over time. It was determined during this review that, because of the complexity of the thyroid system, it is unlikely that current in vitro assays for thyroid toxicity will be able to sufficiently replace in vivo assays for thyroid toxicants. However, the in vitro assays serve an important purpose in providing mode of action information and could provide potential screening tools, and should continue to be developed for use. Moreover, because in vivo assays are added on to preexisting reproductive or developmental screens and tests, there are no additional animals required for the in vivo assays. Specific in vitro assays were identified for development, including the thyroid receptor binding and activation assays, and in vitro assays to evaluate thyroid hormone action. Some in vivo endpoints suggested for further research included neuronal differentiation and migration, measures of histogenesis, and measures for thyroid gland thyroid hormone content, which may be more sensitive indicators of TSH stimulation. The most commonly used endpoints currently used to monitor thyroid function are thyroid hormone levels (T3 and T4), TSH, thyroid gland weight, and thyroid histology. Thyroid endocrinology is rapidly advancing and new discoveries will certainly warrant incorporation into future assays. The development of additional endpoints that measure thyroid hormone's actions peripheral to the HPT axis and the development of new reagents for nonmammalian vertebrate species will significantly improve the ability of today's assays to detect chemicals that disrupt the thyroid system in multiple vertebrate species. It is our hope that this series of thyroid articles will provide regulators and research scientists the information needed for each individual to identify the assays and endpoints most suited for their specific purposes.     

Identification of toxicants in cinnamon-flavored electronic cigarette refill fluids

In a prior study on electronic cigarette (EC) refill fluids, Cinnamon Ceylon was the most cytotoxic of 36 products tested. The purpose of the current study was to determine if high cytotoxicity is a general feature of cinnamon-flavored EC refill fluids and to identify the toxicant(s) in Cinnamon Ceylon. Eight cinnamon-flavored refill fluids, which were screened using the MTT assay, varied in their cytotoxicity with most being cytotoxic. Human embryonic stem cells were generally more sensitive than human adult pulmonary fibroblasts. Most products were highly volatile and produced vapors that impaired survival of cells in adjacent wells. Cinnamaldehyde (CAD), 2-methoxycinnamaldehyde (2MOCA), dipropylene glycol, and vanillin were identified in the cinnamon-flavored refill fluids using gas chromatography–mass spectrometry and high-pressure liquid chromatography (HPLC). When authentic standards of each chemical were tested using the MTT assay, only CAD and 2MOCA were highly cytotoxic. The amount of each chemical in the refill fluids was quantified using HPLC, and cytotoxicity correlated with the amount of CAD/product. Duplicate bottles of the same product were similar, but varied in their concentrations of 2MOCA. These data show that the cinnamon flavorings in refill fluids are linked to cytotoxicity, which could adversely affect EC users.     

Biofluorescent substances for rapid assessment of toxicants on invertebrates

The recently developed 1 h fluorescent test with Daphnia magna was further evaluated. The assay is based on the in vivo observation of enzymatic activity using a fluorogenic substrate that, after cleavage by the corresponding enzyme, releases a fluorescent dye. Influences interfering with the test evaluation were determined and eliminated. It was investigated as to whether new fluorescent tests can be developed for Ceriodaphnia dubia and Hyalella azteca. Also, other fluorescent substrates were tested for their applicability as fluorogenic substrate in combination with D. magna, C. dubia, and H. azteca. © 1996 by John Wiley & Sons, Inc.