Toxics, Volume 6, Issue 3 (September 2018) – 23 articles

Large-scale manufacturing of poly- and perfluorinated compounds in the second half of the 20th century has led to their ubiquity in the environment, and their unique structure has made them persistent contaminants. A recent drinking water advisory level issued by the United States Environmental Protection Agency lowered the advisory level concentration of perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) from 200 nanograms per liter and 400 nanograms per liter, respectively, to 70 nanograms per liter separately or combined. Small temporal variations in PFOS and PFOA concentrations could be the difference between meeting or exceeding the recommended limit. In this study, newly sampled data from a contaminated military site in Alaska and historical data from former Pease Air Force Base were collected. Data were evaluated to determine if monthly variations within PFOS and PFOA existed. No statistically significant temporal trend was observed in the Alaska data, while the results from Pease, although statistically significant, showed the spread of observed contaminant concentrations around the fitted line is broad (as indicated by the low R² values), indicating that collection date has little value in predicting contaminant concentrations. Though not currently the subject of a US EPA health advisory, data on perfluorobutanesulfonic acid (PFBS), perfluorohexane sulfonic acid (PFHxS), perfluoroheptanoic acid (PFHpA), and perfluorononanoic acid (PFNA) were collected for each site and their average concentrations evaluated. Full article

Previous studies have proposed that cadmium (Cd) is a metabolic disruptor, which is associated with insulin resistance, metabolic syndrome, and diabetes. This metal is not considered by international agencies for the study of metabolic diseases. In this study, we investigate the effect of metformin on Cd-exposed Wistar rats at a lowest-observed-adverse-effect level (LOAEL) dose (32.5 ppm) in drinking water. Metabolic complications in the rats exposed to Cd were dysglycemia, insulin resistance, dyslipidemia, dyslipoproteinemia, and imbalance in triglyceride and glycogen storage in the liver, muscle, heart, kidney, and adipose tissue. Meanwhile, rats treated orally with a No-observable-adverse-effect level (NOAEL) dose of metformin (200 mg/kg/day) showed mild improvement on serum lipids, but not on glucose tolerance; in tissues, glycogen storage was improved, but lipid storage was ineffective. In conclusion, metformin as a first-line pharmacological therapy must take into consideration the origin and duration of metabolic disruption, because in this work the NOAEL dose of metformin (200 mg/kg/day) showed a limited efficiency in the metabolic disruption caused by chronic Cd exposure. Full article

Although the increased incidence of type 2 diabetes since the 1950s is thought to be primarily due to coincident alterations in lifestyle factors, another potential contributing factor in industrialized countries is exposure of the population to environmental pollutants and industrial chemicals. Exposure levels of many environmental toxicants have risen in the same time-frame as the disease incidence. Of particular interest in this regard is the metal lead. Although overall lead exposure levels have diminished in recent decades, there is an under-recognized but persistent occurrence of lead exposure in poor underserved urban populations. Although the neural developmental pathologies induced by lead exposures have been well documented, very little is known about the effect of lead exposure on the incidence of chronic metabolic diseases such as type 2 diabetes. Although our understanding of the metabolic health effects of lead exposure is incomplete, there are studies in model systems and a small amount of epidemiological data that together suggest a deleterious effect of environmental lead exposure on metabolic health. This article reviews the human, animal and in vitro studies that have examined the effects of lead exposure on the development of diabetes and related metabolic conditions. Full article

During embryonic development, some hypoxia occurs due to incipient vascularization. Under hypoxic conditions, gene expression is mainly controlled by hypoxia-inducible factor 1 (HIF-1). The activity of this transcription factor can be altered by the exposure to a variety of compounds; among them is cadmium (Cd), a nephrotoxic heavy metal capable of crossing the placenta and reaching fetal kidneys. The goal of the study was to determine Cd effects on HIF-1 on embryonic kidneys. Pregnant Wistar rats were exposed to a mist of isotonic saline solution or CdCl₂ (D_Del = 1.48 mg Cd/kg/day), from gestational day (GD) 8 to 20. Embryonic kidneys were obtained on GD 21 for RNA and protein extraction. Results show that Cd exposure had no effect on HIF-1α and prolyl hydroxylase 2 protein levels, but it reduced HIF-1 DNA-binding ability, which was confirmed by a decrease in vascular endothelial growth factor (VEGF) mRNA levels. In contrast, the protein levels of VEGF were not changed, which suggests the activation of additional regulatory mechanisms of VEGF protein expression to ensure proper kidney development. In conclusion, Cd exposure decreases HIF-1-binding activity, posing a risk on renal fetal development. Full article

Butyrylcholinesterase (BChE) is the evolutionary counterpart to acetylcholinesterase (AChE). Both are expressed early in nervous system development prior to cholinergic synapse formation. The organophosphate pesticide chlorpyrifos (CPF) primarily exerts toxicity through the inhibition of AChE, which results in excess cholinergic stimulation at the synapse. We hypothesized that the inhibition of AChE and BChE by CPF may impair early neurogenesis in neural stem cells (NSCs). To model neurodevelopment in vitro, we used human NSCs derived from induced pluripotent stem cells (iPSCs) with a focus on the initial differentiation mechanisms. Over the six days of NSC differentiation, the BChE activity and mRNA expression significantly increased, while the AChE activity and expression remained unchanged. The CPF treatment (10 μM) caused 82% and 92% inhibition of AChE and BChE, respectively. The CPF exposure had no effect on the cell viability or the expression of the differentiation markers HES5, DCX, or MAP2. However, the shRNA-knockdown of the BChE expression resulted in the decreased or delayed expression of the transcription factors HES5 and HES3. BChE may have a role in the differentiation of NSCs independent of, or in addition to, its enzymatic activity. Full article

Tetramethylenedisulfotetramine (TETS, tetramine) is a toxic organic compound that is used as an effective rodenticide. However, this neurotoxin is not only toxic to rodents, it also causes poisoning in humans. Due to its high level of toxicity for humans, the use of TETS as a rodenticide has been banned and its production has been discontinued. Despite this, human poisoning by this substance is unfortunately still very common. The largest number of poisonings are reported in China, but in the United States, dozens of poisonings still happen annually. TETS is one of the most hazardous pesticides and also a possible chemical warfare agent with no known antidote. In this article, we aim to summarize the biochemical and toxicological data of TETS and hope to cast some light on the toxicological risk to human health. Full article

The present study shows the results of perfuorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA), brominated dioxins (PBDDs) and furans (PBDFs) measured in several marine fish and seafood of commercial interest at different trophic levels of the food chain. The aims were to investigate the level of the contamination in Mediterranean aquatic wildlife, and in farmed fish, to assess human exposure associated to fishery products consumption. Samples of wild fish were collected during three different sampling campaigns in different Food and Agriculture Organization (FAO) 37 areas of the Mediterranean Sea. In addition, farmed fish (gilthead sea bream and European sea bass) from off-shore cages from different marine aquaculture plants. Results showed contamination values of PFOS and PFOA were lower than those detected in sea basins other than the Mediterranean Sea. Concentration values of PFOS were generally higher than those of PFOA; moreover, levels in farmed fish were lower than in wild samples from the Mediterranean Sea. Intake of PFOS and PFOA through fishery products consumption was estimated to be 2.12 and 0.24 ng/kg·BW·day, respectively, for high consumers (95th percentile). Results of 2,3,7,8-substituted congeners of PBDDs and PBDFs were almost all below the limit of detection (LOD), making it difficult to establish the contribution of these compounds to the total contamination of dioxin-like compounds in fish and fishery products. Full article

Seafood is an important component in a healthy diet and may contain methylmercury or other contaminants. It is important to recognize the risks and benefits of consuming seafood. A longitudinal prospective birth cohort study has been conducted to clarify the effects of neurotoxicants on child development—the Tohoku Study of Child Development (TSCD) in Japan. TSCD comprises two cohorts; a polychlorinated biphenyls (PCB) cohort (urban area) and a methylmercury cohort (coastal area). Our previous results from the coastal area showed prenatal methylmercury exposure affected psychomotor development in 18-month-olds, and boys appear to be more vulnerable to the exposure than girls. In this report, we have added the urban area cohort and we reanalyzed the impact of prenatal exposure to methylmercury, which gave the same results as before. These findings suggest prenatal exposure to low levels methylmercury may have adverse effects on child development, especially in boys. Full article

Mercury compounds are known to cause central nervous system disorders; however the detailed molecular mechanisms of their actions remain unclear. Methylmercury increases the expression of several chemokine genes, specifically in the brain, while metallothionein-III (MT-III) has a protective role against various brain diseases. In this study, we investigated the involvement of MT-III in chemokine gene expression changes in response to methylmercury and mercury vapor in the cerebrum and cerebellum of wild-type mice and MT-III null mice. No difference in mercury concentration was observed between the wild-type mice and MT-III null mice in any brain tissue examined. The expression of Ccl3 in the cerebrum and of Cxcl10 in the cerebellum was increased by methylmercury in the MT-III null but not the wild-type mice. The expression of Ccl7 in the cerebellum was increased by mercury vapor in the MT-III null mice but not the wild-type mice. However, the expression of Ccl12 and Cxcl12 was increased in the cerebrum by methylmercury only in the wild-type mice and the expression of Ccl3 in the cerebellum was increased by mercury vapor only in the wild-type mice. These results indicate that MT-III does not affect mercury accumulation in the brain, but that it affects the expression of some chemokine genes in response to mercury compounds. Full article

Methylmercury (MeHg) is a hazardous environmental pollutant, which elicits significant toxicity in humans. The accumulation of MeHg through the daily consumption of large predatory fish poses potential health risks, and the central nervous system (CNS) is the primary target of toxicity. Despite well-described neurobehavioral effects (i.e., motor impairment), the mechanisms of MeHg-induced toxicity are not completely understood. However, several lines of evidence point out the oxidative stress as an important molecular mechanism in MeHg-induced intoxication. Indeed, MeHg is a soft electrophile that preferentially interacts with nucleophilic groups (mainly thiols and selenols) from proteins and low-molecular-weight molecules. Such interaction contributes to the occurrence of oxidative stress, which can produce damage by several interacting mechanisms, impairing the function of various molecules (i.e., proteins, lipids, and nucleic acids), potentially resulting in modulation of different cellular signal transduction pathways. This review summarizes the general aspects regarding the interaction between MeHg with regulators of the antioxidant response system that are rich in thiol and selenol groups such as glutathione (GSH), and the selenoenzymes thioredoxin reductase (TrxR) and glutathione peroxidase (Gpx). A particular attention is directed towards the role of the PI3K/Akt signaling pathway and the nuclear transcription factor NF-E2-related factor 2 (Nrf2) in MeHg-induced redox imbalance. Full article

Several studies have shown the presence of aldehydes (i.e., formaldehyde, acrolein) in mainstream emissions of some e-cigarettes. For this reason, concerns have been raised regarding potential toxicity. The purpose of this research was to measure levels of carbonyls in exhaled breath of e-cigarette users during “vaping” sessions and estimate the respiratory tract (RT) uptake of specific aldehydes, including formaldehyde and acetaldehyde. We measured concentrations of 12 carbonyls in e-cigarette aerosols produced directly by e-cigarettes and in the exhaled breath of 12 participants (19 sessions). Carbonyls were sampled on 2,4-dinitrophenylhydrazine (DNPH) cartridges and analyzed with high performance liquid chromatography (HPLC) coupled with a UV/Vis photodiode detector. We found that in most cases, levels of aldehydes and methyl ethyl ketone (MEK) were significantly higher (2–125 times) in exhaled e-cigarette breaths than in pre-exposed breath. Exposure levels for the most abundant individual carbonyls in e-cigarette emissions—formaldehyde, acetaldehyde, acrolein—were between the limit of quantification (LOQ) and 24.4 μg·puff⁻¹. The mean retention of formaldehyde in the respiratory tract was 99.7 ± 0.9% for all participants, while acetaldehyde retention was 91.6 ± 9.9%. Within the limitation of a small number of participants, our results showed that there is an increase in breath carbonyls during e-cigarette use. Full article

The main chemical forms of mercury are elemental mercury, inorganic divalent mercury, and methylmercury, which are metabolized in different ways and have differing toxic effects in humans. Among the various chemical forms of mercury, methylmercury is known to be particularly neurotoxic, and was identified as the cause of Minamata disease. It bioaccumulates in fish and shellfish via aquatic food webs, and fish and sea mammals at high trophic levels exhibit high mercury concentrations. Most human methylmercury exposure occurs through seafood consumption. Methylmercury easily penetrates the blood-brain barrier and so can affect the nervous system. Fetuses are known to be at particularly high risk of methylmercury exposure. In this review, we summarize the health effects and exposure assessment of methylmercury as follows: (1) methylmercury toxicity, (2) history and background of Minamata disease, (3) methylmercury pollution in the Minamata area according to analyses of preserved umbilical cords, (4) changes in the sex ratio in Minamata area, (5) neuropathology in fetuses, (6) kinetics of methylmercury in fetuses, (7) exposure assessment in fetuses. Full article

The purpose of this updated meta-analysis was to investigate the effect of nitric oxide synthase-3 (NOS3) G894T polymorphisms, air pollution and their interaction on ischemic heart disease (IHD) risk across populations worldwide. Recursive partition trees, nonlinear association curve fit and geographic information system maps were incorporated to verify results of conventional pooled analyses for sources of heterogeneity. Results from 61 studies (16,219 cases, 12,222 controls) revealed a significant increased relative risk (RR) of IHD associated with NOS3 894 polymorphisms TT (RR = 1.44) and GT (RR = 1.37). Subgroup analysis revealed that the TT polymorphism genotype had significantly increased risk of IHD in Caucasian, East Asian, South Asian, and Middle Eastern populations (all p < 0.05). It is important to point out that many countries demonstrated an average risk of greater than two, which identifies the NOS3 894 TT polymorphism as a potential causal factor and biological marker of IHD, based on criteria for strong evidence used in international consensus panels. These 10 countries include Ukraine, the United Kingdom, Brazil, Chile, Japan, South Korea, India, Iran, Egypt and Morocco. For these countries with elevated risk (RR > 2) from the NOS3 894 TT polymorphism, meta-predictive analysis demonstrated an increasing trend in air pollution association with increased NOS3 894 polymorphisms. Further studies are needed to explore the complexity of the associations among NOS3 gene polymorphisms per population stratifications within countries, detailed air pollution data for added specificity for geographic location across time, and disease risk. Full article

Maternal smoking during pregnancy leads to gestational complications and organ disorders in the offspring. As nicotine replacement therapy is often ineffective for smoking cessation, pregnant women turn to alternatives such as heat-not-burn tobacco and e-cigarettes. Recently, the popularly of e-cigarettes has been increasing especially among the youth and pregnant women, mainly due to the advertisements claiming their safety. This has even led to some clinicians recommending their use during pregnancy. E-cigarettes heat e-liquid to produce an aerosol (e-vapor), delivering flavorings and nicotine to the user. However, e-vapor also contains toxins such as formaldehyde along with heavy metals and carcinogenic nitrosamines. In addition, specific flavoring compounds such as diacetyl can be toxic themselves or decompose into toxic compounds such as benzaldehydes. These compounds can induce toxicity, inflammation and oxidative stress in the mothers and can accumulate in the developing fetus, affecting intrauterine development. Recent animal studies suggest that maternal e-vapor exposure during pregnancy could cause respiratory and neurological disorders in the offspring. This review will examine the available literature to shed light on the current understanding of this problem-to-be from lessons learned in animal models. Full article

Lead exposure and a marker of oxidative stress (gamma-glutamyl transferase—GGT), and their effects on life course variables (age, country of birth, education levels, gender, ethnicity, income, and occupation) were explored in this cross-sectional study of United States (U.S.) adults’ ≥ 20 years of age via the National Health and Nutrition Examination Survey (NHANES) 2007–2010 datasets. Country of birth, education levels, gender, ethnicity, income, and occupation showed significant differences depending on the degree of lead exposure, with higher levels of exposure resulting in worse outcomes. Age and GGT were significantly associated with lead exposure. More must be done to mitigate sources of lead exposure, to prevent it from altering the life course of at-risk populations. Full article

Findings from studies of prenatal exposure to pesticides and adverse birth outcomes have been equivocal so far. We examined prenatal exposure to agricultural pesticides in relation to preterm birth and term low birthweight, respectively, in children born between 1998 and 2010, randomly selected from California birth records. We estimated residential exposure to agriculturally applied pesticides within 2 km of residential addresses at birth by pregnancy trimester for 17 individual pesticides and three chemical classes (organophosphates, pyrethroids, and carbamates). Among maternal addresses located within 2 km of any agricultural pesticide application, we identified 24,693 preterm and 220,297 term births, and 4412 term low birthweight and 194,732 term normal birthweight infants. First or second trimester exposure to individual pesticides (e.g., glyphosates, paraquat, imidacloprid) or exposure to 2 or more pesticides in the three chemical classes were associated with a small increase (3–7%) in risk for preterm birth; associations were stronger for female offspring. We did not find associations between term low birthweight and exposure to pesticides other than myclobutanil (OR: 1.11; 95% CI: 1.04–1.20) and possibly the pyrethroids class. Our improved exposure assessment revealed that first and second trimester exposure to pesticides is associated with preterm delivery but is rarely linked with term low birthweight. Full article

Poor water quality has been reported along with a variety of negative health outcomes in the Saharawi refugee camps in Algeria. We assessed the concentration of elements in drinking water and urine in refugee women and children. Twenty-four samples of distributed public drinking water were collected, along with urine samples from 77 women and 296 children. Using inductively coupled plasma mass spectrometry, we analyzed water and urine for 31 and 10 elements, respectively. In addition, the water samples were analyzed for five anions by ion-exchange chromatography. Data were described according to two areas: zone 1 with purified water and water with naturally better quality, and zone 2 with only partially purified water. Most elements in drinking water had significantly higher concentration in zone 2 compared with zone 1. Sodium, chloride, nitrite, and nitrate were the parameters that exceeded the WHO Guidelines for Drinking Water Quality. Among both women and children, urinary concentration of vanadium, arsenic, selenium, lead, iodine, and uranium exceeded reference values, and most of the elements were significantly higher in zone 2 compared to zone 1. Even though water purification in the Saharawi refugee camps has increased during the last years, some elements are still exceeding the WHO guidelines for drinking water quality. Moreover, urinary exposure of some elements exceeded reference values from the literature. Further effort should be made to improve the water quality among the Saharawi refugees. Full article

In 1956 methylmercury poisoning, known as Minamata disease, was discovered among the inhabitants around the Shiranui Sea, Kyushu, Japan. Although about five hundred thousand people living in the area had supposedly been exposed to methylmercury, administrative agencies and research institutes had not performed any subsequent large scale, continuous health examination, so the actual extent of the negative health effects was not clearly documented. In 2009, we performed health surveys in order to examine residents in the polluted area and to research the extent of the polluted area and period of pollution. We analyzed data collected on 973 people (age = 62.3 ± 11.7) who had lived in the polluted area and had eaten the fish there and a control group, consisting of 142 persons (age = 62.0 ± 10.5), most of whom had not lived in the polluted area. Symptoms and neurological signs were statistically more prevalent in the four groups than in the control group and were more prevalent and severe in those who had eaten most fish. The patterns of positive findings of symptoms and neurological findings in the four groups were similar. Our data indicates that Minamata disease had spread outside of the central area and could still be observed recently, almost 50 years after the Chisso Company’s factory had halted the dumping of mercury polluted waste water back in 1968. Full article

After the European Food Safety Authority reviewed reports of methylmercury and heart rate variability (HRV) in 2012, the panel concluded that, although some studies of cardiac autonomy suggested an autonomic effect of methylmercury, the results were inconsistent among studies and the implications for health were unclear. In this study, we reconsider this association by adding a perspective on the physiological context. Cardiovascular rhythmicity is usually studied within different frequency domains of HRV. Three spectral components are usually detected; in humans these are centered at <0.04 Hz, 0.15 Hz (LF), and 0.3 Hz (HF). LF and HF (sympathetic and parasympathetic activities, respectively) are evaluated in terms of frequency and power. By searching PubMed, we identified 13 studies examining the effect of methylmercury exposure on HRV in human populations in the Faroe Islands, the Seychelles and other countries. Considering both reduced HRV and sympathodominant state (i.e., lower HF, higher LF, or higher LF/HF ratio) as autonomic abnormality, eight of them showed the significant association with methylmercury exposure. Five studies failed to demonstrate any significant association. In conclusion, these data suggest that increased methylmercury exposure was consistently associated with autonomic abnormality, though the influence of methylmercury on HRV (e.g., LF) might differ for prenatal and postnatal exposures. The results with HRV should be included in the risk characterization of methylmercury. The HRV parameters calculated by frequency domain analysis appear to be more sensitive to methylmercury exposure than those by time domain analysis. Full article

Background: Benzene is an important component of cigarette smoke and car exhaust. Products containing benzene in concentrations greater than 0.1% are prohibited in Europe, but 1% of benzene is still allowed in gasoline. The purpose of the study was to assess the levels of urine benzene biomarkers in a sample of the general population not occupationally exposed to benzene, resident in the period 2013–2014 in Central Italy, compared to other groups. Methods: The urinary levels of the benzene metabolites S-phenyl-mercapturic acid (SPMA) and cotinine (nicotine metabolite) were determined by means of HPLC with mass spectrometric detection in 1076 subjects. Results: The median SPMA value in smokers was 1.132 µg/g of creatinine while in non-smokers it was 0.097 µg/g of creatinine, and the 95th percentile results were seven times higher. Conclusion: The main source of benzene exposure in the studied population was active smoking, however, non-smokers were also exposed to airborne benzene concentrations. The concentration ranges found in this study can be used as a background reference for occupational exposure assessment to benzene by means of SPMA biomonitoring. Full article

Methylmercury (MeHg) is selectively toxic to the central nervous system, but mechanisms related to its toxicity are poorly understood. In the present study, we identified the chemokine, C-C motif Chemokine Ligand 4 (CCL4), to be selectively upregulated in the brain of MeHg-administered mice. We then investigated the relationship between CCL4 expression and MeHg toxicity using in vivo and in vitro approaches. We confirmed that in C17.2 cells (a mouse neural stem cell line) and the mouse brain, induction of CCL4 expression occurs prior to cytotoxicity caused by MeHg. We also show that the addition of recombinant CCL4 to the culture medium of mouse primary neurons attenuated MeHg toxicity, while knockdown of CCL4 in C17.2 cells resulted in higher MeHg sensitivity compared with control cells. These results suggest that CCL4 is a protective factor against MeHg toxicity and that induction of CCL4 expression is not a result of cytotoxicity by MeHg but is a protective response against MeHg exposure. Full article

There is growing evidence of immunotoxicity related to exposure to toxic trace metals, and an examination of gene expression patterns in peripheral blood samples may provide insights into the potential development of these outcomes. This pilot study aimed to correlate the blood levels of three heavy metals (mercury, cadmium, and lead) with differences in gene expression in 24 participants from the Long Island Study of Seafood Consumption. We measured the peripheral blood mRNA expression of 98 genes that are implicated in stress, toxicity, inflammation, and autoimmunity. We fit multiple linear regression models with multiple testing correction to correlate exposure biomarkers with mRNA abundance. The mean blood Hg in this cohort was 16.1 µg/L, which was nearly three times the Environmental Protection Agency (EPA) reference dose (5.8 µg/L). The levels of the other metals were consistent with those in the general population: the mean Pb was 26.8 µg/L, and the mean Cd was 0.43 µg/L. The expression of three genes was associated with mercury, four were associated with cadmium, and five were associated with lead, although none were significant after multiple testing correction. Little evidence was found to associate metal exposure with mRNA abundance for the tested genes that were associated with stress, toxicity, inflammation, or autoimmunity. Future work should provide a more complete picture of physiological reactions to heavy metal exposure. Full article

Specific gene knockdown facilitated by short interfering RNA (siRNA) is a potential approach for suppressing the expression of ion channels and transporter proteins to kill breast cancer cells. The overexpression of calcium ion channels and transporter genes is seen in the MCF-7 breast cancer cell line. Since naked siRNA is anionic and prone to nuclease-mediated degradation, it has limited permeability across the cationic cell membrane and short systemic half-life, respectively. Carbonate apatite (CA) nanoparticles were formulated, characterized, loaded with a series of siRNAs, and delivered into MCF-7 and 4T1 breast cancer cells to selectively knockdown the respective calcium and magnesium ion channels and transporters. Individual knockdown of TRPC6, TRPM7, TRPM8, SLC41A1, SLC41A2, ORAI1, ORAI3, and ATP2C1 genes showed significant reduction (p < 0.001) in cell viability depending on the cancer cell type. From a variety of combinations of siRNAs, the combination of TRPC6, TRPM8, SLC41A2, and MAGT1 siRNAs delivered via CA produced the greatest cell viability reduction, resulting in a cytotoxicity effect of 57.06 ± 3.72% (p < 0.05) and 59.83 ± 2.309% (p = 0.09) in 4T1 and MCF-7 cell lines, respectively. Some of the combinations were shown to suppress the Akt pathway in Western Blot analysis when compared to the controls. Therefore, CA-siRNA-facilitated gene knockdown in vitro holds a high prospect for deregulating cell proliferation and survival pathways through the modulation of Ca²⁺ signaling in breast cancer cells. Full article