Document Type : Review Paper
1 Department of Toxicology and Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran
2 Iranian Environmental Mutagen Society (IrEMS), Tehran, Iran
Endocrine-disrupting chemicals (EDCs) comprise a huge number of synthetic or natural chemicals with industrial, agricultural, pharmaceutical, cosmetic, and hygienic applications. They mimic the activities of natural hormones through lifetime dietary intakes, inhalational exposures, or other possible routes of administration  . Extensive increase in the incidence and prevalence of non-infectious environmental induced diseases e.g. breast cancer  prostate cancer , infertility  reproductive disorders , congenital abnormalities , neurodegenerative diseases and immune dysfunctions  , could be the consequence of massive use of EDCs over the past 50 years  Fig. 1 describes some of the identifiedhuman health effects of EDCs in both genders.
Most of the agrochemicals (e.g. pesticides and fertilizers) have endocrine-disrupting properties based on their anti-androgenic and/or estrogenic effects  Nowadays massive use of pesticides and fertilizers in the chain of food production/preservation and uncontrolled human exposure to these hazardous chemicals, have become a very big challenge for global health systems. The annual estimated cost of uncontrolled exposure to EDCs was determined as $340 billion (2·33% of GDP) in the United States and €163 billion in European Union (EU) (1·28%) due to health-care costs and lost productivity but this cost estimation is limited to the present generations, not to the intergenerational and trans-generational epigenetic inheritance . The structural diversity of agrochemicals as well as their presence in most environmental sources at undetectable levels by conventional analytical methods, bioaccumulation, biopersitance, and unknown metabolic fate, make the environmental detection and risk assessment of endocrine disrupting agrochemicals more complicated.
Research into nanotechnology applications in the development of novel agrochemicals in the form of “nano fertilizers”, “nano adsorbents” and “nano pesticides”to overcome current health challenges of routine agrochemicals (e.g. organophosphates, carbamates, organochlorines, synthetic pyrethroids) and improvement of crop protection, has become increasingly popular over the past decade. Unfortunately, a large number of risk assessment studies on engineered nanomaterials have characterized the endocrine-disrupting properties for CNTs because they mimic the natural body hormones and interact with hormone receptors in humans and wildlife . Therefore several controversies and key questions address endocrine disrupting properties of CNTs which may soon introduce to future market of novel agrochemicals. This review aims to identify existing knowledge gaps regarding the endocrine-disrupting properties of CNTs and provide directions for future studies in parallel to regulatory activities for the development and promotion of safer nano agrochemicals.
To specify our focus on the role of CNTs on human endocrine disruption through environmental exposures by food, water, and air contamination, all available original and review articles in PubMed were considered for this review using the following keywords for inclusion and exclusion of selected studies.
Carbon nanotube OR CNTs OR Single-Wall Carbon Nanotubes OR SWCNTs OR Multi-Walled Carbon Nanotubes OR Multi-Wall Carbon Nanotube OR MWNT OR MWCNT
Nanoagrochemicals OR Agriculture OR Pesticide OR Nanopesticide OR Fertilizer OR Nano fertilizer OR Endocrine Disruption OR Aryl Hydrocarbon Receptor OR AhR ORLipid metabolism OR Obesity OR Fat accumulation OR Diabetes OR Cardiotoxicity OR Cardiovascular effects OR cardiovascular diseases OR Sexual development OR, Female Reproductive Health OR Teratogenicity OR Teratogen OR Embryotoxicity OREstrogen OR Progesterone OR Testosterone OR Androgen OR FSH OR LH OR Cortisol OR Ovary OR Ovaries OR Uterine, OR vagina OR Hypothalamus–hypophysis axis OR Reproductive cycles OR Pregnancy OR birth OR Male Reproductive Health effects OR Semen quality OR Hypospadias OR Sperm OR Cryptorchidism OR Prostate
Nanocarrier, Drug Delivery, Medicine, Therapy, Protein Detection, cell and organ transplantation,imaging system, Nanodevice, bone tissue regeneration, cancer diagnosis, Vector, scaffold
Applications of Carbon Nanotubes asnano agrochemicals
CNTs are authoritative members of the big family of ENMs based on their special physicochemical properties, a wide range of agrochemical applications, and a rapid surge of industrial attractions in the last decade. CNTs are classified into the following two categories: SWNCTs – Single-Walled Carbon Nanotubes and MWCNTs – Multiple-Walled Carbon Nanotubes. Table 1 shows the capabilities of CNTs’ efficient fertilizer (plant growth inducer ) by increasing the water uptake of plants and enhancing root/shoot lengths and plant dry biomass , . Table 1 shows also the potentials of CNTs as excellent chemicals for the detection and remediation of environmental pollutants e.g. heavy metals, persistent organic pollutants (POPs) especially organochlorine pesticides, dyes, the residue of pharmaceuticals and EDCs based on exhibiting strong adsorptive properties  and their antimicrobial and antifungal properties.
Environmental risks and benefits of CNTs applications
Due to the highly porous and hollow structure of CNTs, large specific surface area, excellent adsorptive capacities as well as their short equilibrium times and strong interaction between CNTs and pollutant molecules, these highly available nanomaterials, have been gradually applied for the removal of organic contaminants with endocrine-disrupting effects from wastewater through adsorption process as novel absorbent . Soluble carbon nanotubes (CNTs) have shown promising roles as adsorptive materials against Endocrine-disrupting chemicals(EDCs)such as Bisphenol A (BPA) but the adsorptive capabilities of CNTs to EDCs may change the final toxic properties of the possible complexes in human and other living organisms and cause reproductive toxicity.
Besides the above remarkable effects of CNTs in agriculture and remediation, CNTs provide toxic effects on plants by inducing reactive oxygen species (ROS) leading to abnormal cell death. Unusual accumulation of CNTs in soils can cause hazardous effects on soil microbial population, diversity, and composition. They can modify the balance between plant-toxic metals in soil and accelerate the translocation of heavy metals and metalloids into the plant tissues. . CNTs release into water and wastewater treatment systems when it is used as an adsorbent for water and soil treatment . Studies on adsorptive properties of SWCNTs indicate that SWCNTs effectively adsorbed 17β-estradiol and natural hormones in animals and plants which cause hormonal deficiencies . Increasing environmental levels of MWCNTs is dangerous due to their strong affinities to estrogenic compounds ( 17β-estradiol) in aquatic systems. Hormonal affinities of MWCNTs cause definite alterations in estrogenic responses to other EDCs by increasing their bioavailability.It seems that other harmful chemicals would be able to bind and activate soluble estrogen receptors (ERs) and make more critical situations for determining the potential health risks of CNTs alone in biological systems.
Toxicokinetic and toxicodynamic of CNTs as strong endocrine disruptors
3-1Biodistribution: Environmental induced toxic effects of CNTs are highly dependent on the route of exposure which usually happens through the ingestion of possible residues in food resources or inhalation of contaminated air during occupational activities. The toxicity of CNTs, biodistribution, bioaccumulation, and target organ toxicity are complex subjects and mostly related to the structure and physicochemical properties. The number of walls, chirality, diameter and length , purity, production method and CNT functionalization  fibrogenic properties, hydrophobicity, high surface area and biopersistence  is the most important CNTs characteristics that may cause organ toxicity and systemic adverse health in the endocrine system but among mentioned variables, the quality of functionalization is the most important factor which may change the future of CNTs in the body . Covalently functionalized CNTs tend to be excreted through urine, whereas pristine and non-covalently functionalized CNTs tend to accumulate in spleen and liver of exposed organisms and these types of bioaccumulation in liver and spleen may cause short term or long term toxic responses and disease development .
Metabolism: After total body intake and making biological effective concentrations of CNTs, some members especially SWCNTs isoforms could act as competitive inhibitors of CYP3A4, CYP3A5, and CYP2D6 and these enzyme inhibitions may potentiate the endocrine-disrupting properties of other xenobiotics in co-exposure models. Computational and animal models showed the interaction of CNTs with CYP3A4, a critical and high abundance drug-metabolizing cytochrome P450 enzyme .
3-3Hormonal effects: Dose-dependent inhi-bition of CYP3A4 by CNTs mediates the conversion of testosterone (male steroid hormone) to its major metabolite, 6β-hydroxy testosterone and finally cause male hormonal dysregulation, interfere with the metabolism of other xenobiotics and provides a molecular mechanism for toxic responses .Binding assays indicate the binding capacities of SWCNTs to Estrogen receptors (ER) as one of the most important receptors of human reproductive system .Interacting of SWCNTs to estrogen receptor alpha in a very low concentration range ( 26.43 - 259.01 pg/ml) could be considered as a very critical mechanism and the main molecular initiating event leading to endocrine and reproductive toxicity of SWCNTs . Interactions of CNTs with gonadotropins is another key mechanism for endocrine-disrupting effects of CNTs.Strong affinities between blood glycoproteins and CNTs have been described by molecular and experimental studies. Molecular dynamics, structure, and free binding energy of human Follicle Stimulating Hormone ( FSH) on the surface of SWCNT causes that human FSH in aqueous solution strongly adsorbs onto SWCNT and this strong interaction could change the hormonal activity of FSH, causes dysregulations in hypothalamus-hypophysis axis and endocrine and reproductive toxicity . The same interaction could be predictable between other types of CNTs and circulating hormones in the body based on the mentioned physicochemical properties.
Effects on ovaries: The effects of MWCNTs on ovarian function and granulosa cell steroidogenesis showed the inhibitory role of MWCNTs with different lengths on progesterone secretion and the expression of steroidogenic acute regulatory protein based on cytotoxicity, oxidative stress and mitochondria damages mechanisms .
CONTRIBUTION ON CNTS IN THE PATH-OGENESIS OF OBESITY, DIABETES, AND CARDIOVASCULAR EFFECTS
Lipid metabolism and obesogenic effects of CNTs
Because of the global increase in the prevalence of obesity  and its related syndromes e.g. nonalcoholic fatty liver disease (NAFLD)  and metabolic syndrome , the metabolic and obesogenic effects of all chemicals and nanomaterials should be concerned by health regulatory agencies before any official approval. For the first time, one study points towards the cardiovascular risks of MWCNTs through inhalational exposures and showed the role of a single intratracheal instillation of MWCNTs in a dose range of 0, 18, 54 or 162μg/mouse on lipid profile of female C57BL/6 mice. Treated animals showed a significant increase in plasma total cholesterol, low-density/very low-density lipoprotein (LDL, VLDL), APR proteins, SAA3 and haptoglobin, and histopathological studies showed abnormal changes in liver morphology following exposure to MWCNTs with different physicochemical properties. The results of this study link the importance of MWCNTs pulmonary exposure with abnormal body weight gain, changes in lipid metabolism, and increased risk of cardiovascular disease .
Another parallel study at the same year (2015) revealed the role of intratracheal exposure to two different MWCNTs on development of nonalcoholic steatohepatitis (NASH)-like phenotype, chara-cterized by inflammation, hepatic steatosis, and fibrosis as well as NASH-like phenotype which was consistent with up-regulation of interleukin 6 (IL-6) and plasminogen activator inhibitor-1 (PAI-1). Other abnormalities including overexpression of NF-κB, p65, impaired cholesterol homeostasis, and suppression of peroxisome proliferator-activated receptor-gamma (PPARγ) in the hepatocytes of exposed animals were also detected . The next study confirmed the aggravating role of MWCNTs in nonalcoholic steatohepatitis in Sprague Dawley rats by inducing oxidative injury .
Diabetogenic effects of CNTs
The growing use of carbon nanotubes (CNTs) in agrochemicals, emphasizes the importance of studies on biocompatibility evaluation and specific toxic effects of CNTs in the pancreas. In silico studies and computational analysis have provided insights into the toxic response of CNTs which causes Type 2 Diabetes Mellitus (T2DM) because CNTs could be recognized as pathogens by the Toll-like receptors that may induce the expression of inflammatory secretory proteins . A study in three months old BALB/c mice that exposed to CNTs via injectional rout showed the pancreatic uptake of CNTs but the pancreas remained histologically normal, with no tissue damage, inflammatory infiltrate or inorganic deposits despite inducing hepatic, renal, pulmonary and spleen tissue damages .One other study on the effects of SWCNTs on islets and β-cells, demonstrated decreased viability of islets cells in a dose-dependent manner by overproduction of reactive oxygen species (ROS) and raise of oxidative stress biomarkers including activities of superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA);and glutathione (GSH) peroxidase (GSH-Px); and content of GSH and mitochondrial membrane potential (MMP)  Direct diabetogenic effects of SWCNTs and MWCNTs should be considered as important subjects for further preclinical evaluations in realistic models by different routes of administration.
Cardiovascular effects CNTs
There are several primary mechanisms to link CNTs’ exposure to cardiovascular toxicity. The first is their inflammatory and oxidative properties, direct particle interactions based on their excellent systemic absorption, bioreactivity, and their capacities for neural and hormonal alterations  . Oral administration of SWCNTs could induce cardiovascular diseases in mice and rats after excellent GI absorption and distribution throughout most of the body organs including the liver, lungs, brain, and spleen by inducing oxidative stress and eliminating through kidney and bile duct . SWCNTs induced oxidative stress has investigated rat aortic endothelial cells (RAECs) and showed cellular, DNA, and protein damages and oxidative damage following SWCNT exposure which may result in the progression of many serious diseases especially cardiovascular abnormalities .
The potential cardiotoxicity of MWCNT has not been elucidated yet but an initial study demonstrated the bioreactivity of MWCNT by increasing cell permeability in human microvascular endothelial cells (HMVEC). This toxic effect was mediated by reactive oxygen species (ROS) production, actin filament remodeling, and promoting cell migration in HMVEC. Studies supported the role of MWCNT on elevating the levels of monocyte chemoattractant protein-1 (MCP-1) and intercellular adhesion molecule 1 (ICAM-1) in HMVEC and elucidated the potential human toxicity of MWCNT at the cellular level . Male C57BL/6J mice, exposed to single doses of three different forms of MWCNTat doses of 0.01 - 100 μg showed myocardial ischemia/reperfusion injuries based on the form of administered MWCNTs .Another study in 2014, confirmed previous findings regarding the cardiotoxic effects of MWCNTs following single intratracheal instillation of 1, 10 or 100 μg MWCNT in Sprague-Dawley rats inLangendorff isolated heart model. Increased endothelin-1 (ET-1) release and depression of coronary flow during early reperfusion were observed in exposed rats and the promoting role of MWCNTs on cardiac injury and depressed coronary flow by invoking vasoconstrictive mechanisms involving ET-1, thromboxane, Rho-kinase, and cyclooxygenase was discovered . One more study also indicated that exposure to MWCNT increases the adherence of monocytes onto the endothelium, elevates the levels of oxidative stress-mediated transformation of monocytes to foam cells which are closely correlated with accelerated progression of atherosclerosis .
Later studies in 2015 showed the cardiovascular toxicity of long-term exposure to MWCNTs particularly in occupationally exposed workers with preexisting cardiovascular disorders. In this study, four different MWCNTs with different iron contents and length caused a persistent decrease in the heart rate of spontaneously hypertensive (SH) rats by inducing sustain inflammation of the lung and heart of animals as well as morphological lesions after 30 days repeated dose exposures by intratracheal instillation Another study in mice model of atherosclerosis showed that pharyngeal aspiration of 40 μg MWCNT, once a week for 16 consecutive weeks to female apolipoprotein E-deficient (apo E-/-) mice, elevates the levels of total protein and lactate dehydrogenase (LDH), surfactant protein-D, and mucin without any markedly effect on plasma cholesterol levels  These in vivo and epidemiological evidence suggest the cardiotoxicity of both types of CNTs and increased risk of myocardial ischemia and atherosclerosis in repeated dose and long term exposures.
DEVELOPMENTAL AND REPRODUCTIVE TOXICITY OF CNTS
The underlying mechanisms of CNTs’ hormonal activities reviewed in the last sections of this paper. As described in Table 2, MWCNTs are embryotoxic in rodent and aquatic models. Collected data show the dose/concentration-dependent developmental toxicity of MWCNTs and SWCNTs with different physicochemical properties in acute and repeated models by inducing early and late resorption of the fetus, decreased fetal weights, fetal malformations, fetal death in rodent and aquatic models.
CONCLUSION AND PERSPECTIVES
Despite restricted, conflicted, and inconclusive body of information concerning the real concentrations and behavior of CNTs in vitro and in vivo models and lack of epidemiological studies on CNTs, present evidence suggests the prominent role of CNTs in the pathogenesis of endocrine and reproductive disorders. The fact that CNTs can disrupt the endocrine and reproductive system, which may eventually lead to cardiovascular toxicity, obesity, changes in lipid metabolism, has gathered support from several in vitro and in vivo which described in the present review.This review showed the underlying mechanism following the interference of EDCs with the body’s endocrine system which induces structural and functional abnormalities in gonads and increases the risk of adverse health effects. Differential adverse health effects of CNTs start usually after excellent systemic absorption through direct binding to circulating hormones or hormone receptors, alteration of hormonal activity and synthesis and changing the biological responses of other EDCs therefore beside available data about the CNTs interactions with various cells, tissues, endocrine organs, and organ systems and whole organs as a prerequisite for safety evaluations, special attention to their endocrine-disrupting properties for registered new CNTs based agrochemicals as new candidates for agricultural, medical and industrial applications. This review to show a new picture from CNTs which could be more complicated than described because the wide range of CNTs with different physicochemical properties and unrecognized conformational changes (e.g. agglomeration) with potential impact on bioavailability and endocrine toxicity may cause unpredicted toxicities in human exposure based on their background factors e.g. nutrition, drugs, occupation, and lifestyle factors moreover any Chemical change in CNTs (functionalization) or conformational alteration (agglomeration) can significantly influence their toxicity potential and their receptor interactions.
Apart from these knowledge gaps, some initial properties of CNTs, relevant for their interactions with plants during agricultural applications have been identified and explained in this review. Exposure of plants with CNTs is frequently associated also with internal translocation of CNTs and some systemic effects because they can be taken up by herbs and their absorbed fractions can induce systemic physiological responses. Low plant tissue levels of CNTs (<100 mg L−1) could have beneficial e.g. improved seed germination, hormesis, or lack of toxicity but higher external concentrations usually cause inhibitory effects on plant growth and differentiation. Formation of reactive Oxygen Species (ROS) and oxidative stress are the main common toxic mechanisms of CNTs in plants and animals but despite indicated facts, CNTs have been proposed as the main engineered nanomaterials for developing next generation of nano agrochemicals by industries.
Since the discovery of the first generation of synthetic pesticides during World War II, numerous chemicals have been developed and applied as an insecticide, rodenticide, fungicide, herbicide, fumigants, and disinfectants. Despite overproduction and large scale application of conventional pesticides, a wide variety of them have become practically inefficient because they were unable to reach the specific pests or the targeted strains may become resistant against them . In parallel with the production of old and new molecules, undetermined cases of acute pesticide poisoning (APP), and countless human and animal death via indirect or indirect poisoning through systemic absorption of pesticide residues in contaminated water, air, and food or by occupational routs have happened. Is it possible to have similar tragedies with CNTs as novel agrochemicals in the next decades?
Although the production and application of highly toxic pesticides banned in many countries, widespread use and lack of standardized case definition, risk assessment, risk management, and regulation globally, led to the pollution of ecosystems, a decline in populations of insect pollinators, and increased risk of pesticide-induced human diseases. However, a surprisingly restricted, conflicted, and inconclusive body of information exists concerning the real concentrations and behavior of these materials in biological systems especially in humans and their interactions with various cells, tissues, organs, organ systems, and whole organs as a prerequisite for safety evaluations. Now the critical question is about environmental and human safety regarding their future wide range of applications, their residues in environmental “ Is humanity waiting for a similar or even worse scenario with CNTs as the new generation of agrochemicals for the next decades? " In an optimistic scenario, applications of MWCNTs and SWCNTs in the agriculture sector showing potential impacts on the endocrine system and their efficacy in improvement of plant growth as nano fertilizer, absorption of pollutants as nano adsorbents or pes control as nano pesticides are at a very nascent stage and more risk assessment studies are necessary for their future applications. Evaluation of endocrine disrupting properties of CNTs should be considered as an essential step in risk assessment and management of CNTs through in vivo and in vitro studies using valid biomarkers to emphasize their roles of endocrine systems and hormone-dependent although these models are not always valid or enough for human risk evaluations this is a critical, initial and mandatory step for regulating all new chemicals for health, agricultural or industrial applications especially CNTs which described in this review.
CONFLICT OF INTEREST
Author declares no conflict of interest.
Apolipoprotein E-deficient mice apoE-/-mice
Acute-phase proteins/ acute-phase
reactant (APR) APR proteins
Carbon Nanotubes CNTs
Cytochrome P450 enzyme Cyp450/CYP
Endocrine-disrupting chemicals EDCs
Engineered Nanomaterials ENMs
Follicle Stimulating Hormone FSH
Human microvascular endothelial cells HMVEC
Glutathione peroxidase GSH-Px
Interleukin 6 IL-6
Intercellular adhesion molecule 1 ICAM-1
GDP Gross Domestic Product
Single-Wall Carbon Nanotubes SWCNTs
Monocyte chemoattractant protein-1 MCP-1
Multi-Walled Carbon Nanotubes MWCNT
Nonalcoholic steatohepatitis NASH
Nonalcoholic fatty liver disease NAFLD
Reactive Oxygen Species ROS
Serum amyloid A 3 SAA3
Type 2 Diabetes Mellitus T2DM
Low-density lipoprotein LDL
Luteinizing Hormone LH
Mitochondrial membrane potentialMMP
Plasminogen activator inhibitorPAI-1
Peroxisome proliferator-activated receptor-gamma PPARγ
Rat aortic endothelial cellsRAECs
Spontaneously hypertensive rats SH rats
Superoxide dismutase SOD
Nuclear factor-kappa B NF-κB
Very low-density lipoprotein VLDL