The role of NF-κB and AhR transcription factors in lead-induced lung toxicity in human lung cancer A549 cells.

Lead (Pb) is recognized as the first heavy metal of the top six toxic air pollutants threatening human health and the second hazardous substance. Pb exposure is associated with lung impairment and high incidences of lung cancer. Nuclear factor kappa B (NF-κB) and aryl hydrocarbon receptor (AhR) signaling pathways are known to be expressed and play an important role in the lung. However, the link between Pb lung toxicity and NF-κB and/or AhR pathways remains unclear. This study was established to explore the role of NF-κB and AhR modulation in Pb-induced lung toxicity in human lung cancer A549 cells. In the current study, treatment of A549 cells with Pb significantly induced cell apoptosis as evidenced by increasing a) the percentage of cells underwent apoptosis determined by flow cytometry and b) p53 mRNA level. Pb treatment induced oxidative stress by a) increasing the formation of reactive oxygen species and b) decreasing GSTA1 mRNA levels. The toxic effects of Pb on the lung was associated with significant increases in NF-κB and AhR levels which was accompanied with increases in downstream targets genes, iNOS and CYP1A1, respectively. Inhibition of NF-κB or AhR either chemically using resveratrol or genetically using small interfering RNA (siRNA) significantly rescued A549 cells from Pb-mediated lung toxicity. The results clearly indicate that Pb-mediated lung toxicities are NF-κB and AhR-dependent mechanism.

Genotoxic impact of long-term cigarette and waterpipe smoking on DNA damage and oxidative stress in healthy subjects.

Although a plethora of studies have examined tobacco smoke-cancer disease association, the involvement of cellular genetic toxicity remains unclear. Therefore, the present study provides molecular evidence for a pathway involved in the DNA damage induced by long-term cigarette and waterpipe smoke in human subjects. The study population consisted of 45 subjects who were divided into three groups; healthy nonsmokers group, cigarette smokers group, and waterpipe smokers group. A questionnaire and consent form was distributed and signed by all participants. Total RNA was extracted from the blood using PAXgene Blood RNA Kit and mRNA expression levels of target genes were quantified by RT-PCR. Our results showed that 80% of the participants smoke 20-39 cigarettes/day, whereas 12% smoke more than 40 cigarettes/day. With regard to waterpipe smoke, the majority (46%) smoke more than 5 times/week. Both cigarette and waterpipe smokers showed increased the plasma levels 8-hydroxy-2'-deoxyguanosine (8-OHdG), of DNA damage marker. In addition, the mRNA expression levels of DNA repair genes (OGG1 and XRCC1) were significantly inhibited in both cigarette and waterpipe smokers groups by 30% and 60%, respectively. This was associated with a marked decrease (50%) in the expression of detoxifying genes (NQO1 and GSTA1) with an increase in CYP1A1 mRNA expression, a cancer-activating gene. Both cigarette and waterpipe smokers increased in the plasma concentrations of several toxic heavy metals such as Cd (130%), Pb (47%), and Ni (30%). In conclusion: the present findings clearly explore the genotoxic effect of cigarette and waterpipe smoking on human DNA.

Cathine and cathinone disposition kinetics and neurotransmitter profile in several organs of rats exposed to a single dose of Catha edulis (Vahl) Forssk. ex Endl. extract.

OBJECTIVES: Catha edulis (Vahl) Forssk. ex Endl. (Khat) is a stimulant plant that contains cathine and cathinone, which its abuses induce euphoria, alertness, and motor activity. Since the toxicokinetics of these substances remain unclear, this study was carried out to investigate the disposition kinetics of cathine and cathinone, the neurotransmitter profile, following a single dose of C. edulis extract in rats. METHODS: Twenty-four adult male Wistar albino rats (250-300 g) were randomly selected and divided into six groups of four rats each. All groups received a single oral dose of 2,000 mg/kg body weight, and blood and tissue samples from the brain, lung, heart, liver, and kidney were obtained at intervals of 0.5, 1, 2.5, 5, 12, and 24 h. The cathine and cathinone concentrations were identified and quantified using ion trap ultra-high performance liquid chromatography (HPLC-IT/MS). The neurotransmitter profile was detected using the quadrupole time of flight UPLC-QTOF/MS method. RESULTS: The lung, liver, and heart tissues attained the highest levels of cathine, while the highest level of cathinone was determined in the heart. Cathine and cathinone concentrations in the blood and heart peaked at 0.5 h. The concentrations peaked in the brain 2.5 h later, indicating that the heart had an immediate effect, whereas the brain had a longer-lasting one. They have longer half-lives (2.68 and 5.07 h, respectively) and may remain in the brain for longer durations (3.31 and 2.31 h, respectively). The neurotransmitters epinephrine, dopamine, norepinephrine, and serotonin were detected in a delayed, prolonged and organ-specific manner. CONCLUSIONS: Cathine and cathinone were deposited in considerable concentrations in all tissues analyzed, with the highest Cmax in the lung and Tmax in the heart tissues but not in the brain. In addition, neurotransmitters such as adrenaline, dopamine, norepinephrine, and serotonin were differentially detected in all tested samples in a organ-specific fashion. More study is needed to identify cathine and cathinone's effects on neurotransmitter profiles. Nevertheless, these findings provided a further basis for experimental, clinical, and forensic investigations.

Lead Nitrate Induces Inflammation and Apoptosis in Rat Lungs Through the Activation of NF-κB and AhR Signaling Pathways.

Lead (Pb) is one of the most frequent hazardous air contaminants, where the lungs are particularly vulnerable to its toxicity. However, the Pb distribution and its impact on lung inflammation/apoptosis and particularly the involvement of nuclear factor kappa B (NF-κB) and aryl hydrocarbon receptor (AhR) signaling pathways in Pb-induced lung toxicity have not yet been fully investigated. Adult male Wistar albino rats were exposed to Pb nitrate 25, 50, and 100 mg/kg b.w. orally for 3 days. The histopathological changes of several rat organs were analyzed using hematoxylin and eosin staining. The concentrations of Pb ion in different organ tissues were quantified using inductive coupled plasma mass spectrometry, while gas chromatography-mass spectrometry was used to identify organic compounds. The changes in the mRNA and protein expression levels of inflammatory and apoptotic genes in response to Pb exposure were quantified by using RT-PCR and Western blot analyses, respectively. Treatment of rats with Pb for three consecutive days significantly increased the accumulation of Pb in lung tissues causing severe interstitial inflammation. Pb treatment also increased the percentage of lung apoptotic cells and modulated apoptotic genes (Bc2, p53, and TGF-α), inflammatory markers (IL-4, IL-10, TNF-α), and oxidative stress biomarkers (iNOS, CYP1A1, EphX) in rat lung tissues. These effects were associated with a significant increase in organic compounds, such as 3-nitrotyrosine and myeloperoxidase, and some inorganic elements, such as selenium. Importantly, the Pb-induced lung inflammation and apoptosis were associated with a proportional increase in the expression of NF-κB and AhR mRNAs and proteins. These findings clearly show that Pb induces severe inflammation and apoptosis in rat lungs and suggest that NF-κB and AhR may play a role in Pb-induced lung toxicity.

Habitual khat chewing alters urinary inorganic profile in adult healthy males.

OBJECTIVES: Khat (Catha edulis) is a stimulant plant, and it is abusive to induce euphoria, alertness and activity. Concomitant use of medications and khat chewing predisposes to the appearance of drug interactions result in treatment failure or toxicity. This study determined the changes in the urinary inorganic profile in adult healthy males who are chewing khat compared with non-khat chewer males. METHODS: A total of 40 adult non-smoker healthy males (20 khat chewer and 20 non-khat chewer) aged 24-30 years were selected. Khat chewer samples were positive for cathinone and cathine and negative for other drug of abuse, while non-khat chewer samples were negative for drug of abuse include cathinone and cathine. Samples were selected according to their results in immunoassay and gas chromatography mass spectrometry (GCMS) analysis. Cathine and cathinone were confirmed using liquid chromatography mass spectrometry (LC-MSMS) analysis. Inorganic profile includes titanium (Ti), cobalt (Co), copper (Cu), zinc (Zn), cadmium (Cd), and lead (Pb) were determined by using inductively coupled plasma-mass spectrometry (ICP-MS). RESULTS: The levels of Ti, Co, Zn, Cd, and Pb in urine were significantly higher among the khat chewer group compared with non-khat chewer. Ti, Cd, Co, Pb and Zn urine levels were 0.5-, 1.5-, 1.15-, 5-, and 8.2-fold higher in the khat chewer group compared to non-khat chewer, respectively. CONCLUSIONS: We suggested that continuous khat chewing has a long term effect on metabolic pathway of therapeutic drugs that result in toxicity or failure of therapy.

Habitual khat chewing alters urinary inorganic profile in adult healthy males.

OBJECTIVES: Khat (Catha edulis) is a stimulant plant, and it is abusive to induce euphoria, alertness and activity. Concomitant use of medications and khat chewing predisposes to the appearance of drug interactions result in treatment failure or toxicity. This study determined the changes in the urinary inorganic profile in adult healthy males who are chewing khat compared with non-khat chewer males. METHODS: A total of 40 adult non-smoker healthy males (20 khat chewer and 20 non-khat chewer) aged 24-30 years were selected. Khat chewer samples were positive for cathinone and cathine and negative for other drug of abuse, while non-khat chewer samples were negative for drug of abuse include cathinone and cathine. Samples were selected according to their results in immunoassay and gas chromatography mass spectrometry (GCMS) analysis. Cathine and cathinone were confirmed using liquid chromatography mass spectrometry (LC-MSMS) analysis. Inorganic profile includes titanium (Ti), cobalt (Co), copper (Cu), zinc (Zn), cadmium (Cd), and lead (Pb) were determined by using inductively coupled plasma-mass spectrometry (ICP-MS). RESULTS: The levels of Ti, Co, Zn, Cd, and Pb in urine were significantly higher among the khat chewer group compared with non-khat chewer. Ti, Cd, Co, Pb and Zn urine levels were 0.5-, 1.5-, 1.15-, 5-, and 8.2-fold higher in the khat chewer group compared to non-khat chewer, respectively. CONCLUSIONS: We suggested that continuous khat chewing has a long term effect on metabolic pathway of therapeutic drugs that result in toxicity or failure of therapy.

Drug-Related Problems and Pharmacist Intervention at a General Hospital in the Jazan Region, Saudi Arabia.

OBJECTIVE: This study examined the incidence of drug-related problems (DRPs) in different inpatient departments along with the medical team response to pharmacist's action in addressing DRPs at Jazan Hospital, Saudi Arabia. PATIENTS AND METHODS: This retrospective study was conducted among inpatients at Ministry of Health hospital in Jazan, a region in southwestern Saudi Arabia. We collected data for a 2-year period (from 2016 to 2017). For any detected DRP of the ordered medications for dispensing, the inpatient pharmacist is sending report for that particular DRP with recommendation to the medical team which in turn might accept or reject such recommendation. Serious drug-drug interactions, as part of DRP, were assessed by utilizing three different online DDI checkers, including Lexi-Comp, Medscape, and Drugs.com. RESULTS: The most common type of DRP was serious drug-drug interactions (49%). Over the study period, most incidences of DRPs were decreased. Of the DRPs in 2016 and 2017, antibiotics were the most commonly involved (51% and 69.5%) of cases, respectively, followed by proton pump inhibitors (25.3% and 14.1%) and statins (12.9% and 9.4%). Interestingly, of the 241 interventions for DRPs in 2016, 199 (82.5%) were accepted, resulting in a change in drug therapy (p=0.006). In 2017, 90 (70.3%) interventions out of 128 were accepted by the physician and the drug therapy changed (p=0.029). CONCLUSION: Pharmacist interventions appear to decrease the incidence of DRPs, which emphasize the importance of an optimal pharmaceutical care plan for clinical care settings.

BCL-2 Inhibitor Venetoclax Induces Autophagy-Associated Cell Death, Cell Cycle Arrest, and Apoptosis in Human Breast Cancer Cells.

INTRODUCTION: Venetoclax (VCX) is a selective BCL-2 inhibitor approved for the treatment of leukemia and lymphoma. However, the mechanisms of anti-cancer effect of VCX either as a monotherapy or in combination with other chemotherapeutic agents against breast cancer need investigation. METHODS: Breast cancer cell lines with different molecular subtypes (MDA-MB-231, MCF-7, and SKBR-3) were treated with different concentrations of VCX for indicated time points. The expression of cell proliferative, apoptotic, and autophagy genes was determined by qRT-PCR and Western blot analyses. In addition, the percentage of MDA-MB-231 cells underwent apoptosis, expressed higher oxidative stress levels, and the changes in the cell cycle phases were determined by flow cytometry. RESULTS: Treatment of human breast cancer cells with increasing concentrations of VCX caused a significant decrease in cells growth and proliferation. This effect was associated with a significant increase in the percentage of apoptotic MDA-MB-231 cells and in the expression of the apoptotic genes, caspase 3, caspase 7, and BAX, with inhibition of anti-apoptotic gene, BCL-2 levels. Induction of apoptosis by VCX treatment induced cell cycle arrest at G0/G1 phase with inhibition of cell proliferator genes, cyclin D1 and E2F1. Furthermore, VCX treatment increased the formation of reactive oxygen species and the expression level of autophagy markers, Beclin 1 and LC3-II. Importantly, these cellular changes by VCX increased the chemo-sensitivity of MDA-MB-231 cells to doxorubicin. DISCUSSION: The present study explores the molecular mechanisms of VCX-mediated inhibitory effects on the growth and proliferation of TNBC MDA-MB-231 cells through the induction of apoptosis, cell cycle arrest, and autophagy. The study also explores the role of BCL-2 as a novel targeted therapy for breast cancer.

Gefitinib loaded nanostructured lipid carriers: characterization, evaluation and anti-human colon cancer activity in vitro.

NLC containing Gefitinib (NANOGEF) was prepared using stearic acid, sesame oil and surfactants (sodium lauryl sulfate and tween 80). NANOGEFs were evaluated for particle size, polydispersity index (PdI), zeta potential, entrapment efficiency (EE), stability, release studies and cytotoxicity studies (MTT assay). The optimized NANOGEF exhibited particle size of 74.06 ± 9.73 d.nm, PdI of 0.339 ± 0.029 and EE of 99.76 ± 0.015%. The TEM study revealed spherical shape of NANOGEF formulations. The slow and sustained release behavior was exhibited by all NANOGEFs. The effects of surfactants were observed not only on particle size but also on zeta potential, entrapment efficiency, stability and release studies. The MTT assay revealed 4.5 times increase in cytotoxicity for optimized NANOGEF (IC50 = 4.642 µM) when compared with Gefitinib alone (IC50 = 20.88 µM in HCT-116 cells). Thus NANOGEF may be considered as a potential drug delivery system for the cure of colon cancer.

Gene expression profiling to identify the toxicities and potentially relevant human disease outcomes associated with environmental heavy metal exposure.

Heavy metals are the most commonly encountered toxic substances that increase susceptibility to various diseases after prolonged exposure. We have previously shown that healthy volunteers living near a mining area had significant contamination with heavy metals associated with significant changes in the expression of some detoxifying genes, xenobiotic metabolizing enzymes, and DNA repair genes. However, alterations of most of the molecular target genes associated with diseases are still unknown. Thus, the aims of this study were to (a) evaluate the gene expression profile and (b) identify the toxicities and potentially relevant human disease outcomes associated with long-term human exposure to environmental heavy metals in mining area using microarray analysis. For this purpose, 40 healthy male volunteers who were residents of a heavy metal-polluted area (Mahd Al-Dhahab city, Saudi Arabia) and 20 healthy male volunteers who were residents of a non-heavy metal-polluted area were included in the study. Total RNA was isolated from whole blood using PAXgene Blood RNA tubes and then reversed transcribed and hybridized to the gene array using the Affymetrix U219 GeneChip. Microarray analysis showed about 2129 genes were identified and differentially altered, among which a shared set of 425 genes was differentially expressed in the heavy metal-exposed groups. Ingenuity pathway analysis revealed that the most altered gene-regulated diseases in heavy metal-exposed groups included hematological and developmental disorders and mostly renal and urological diseases. Quantitative real-time polymerase chain reaction closely matched the microarray data for some genes tested. Importantly, changes in gene-related diseases were attributed to alterations in the genes encoded for protein synthesis. Renal and urological diseases were the diseases that were most frequently associated with the heavy metal-exposed group. Therefore, there is a need for further studies to validate these genes, which could be used as early biomarkers to prevent renal injury.

Differential Effects of Sunitinib on the Expression Profiles of Xenobiotic-Metabolizing Enzymes and Transporters in Rat Liver and Kidneys.

Sunitinib (SUN) is a multi-targeted tyrosine kinase inhibitor that was recently approved for the treatment of gastrointestinal tract and renal cancers. To date, very little is known about the effects of SUN on the expression of hepatic and renal xenobiotic-metabolizing enzymes (XMEs) and transporters. The present study was designed to investigate the capacity of chronic SUN treatment to modulate the mRNA and protein expression levels of phase I cytochrome P450 (CYP), phase II conjugating enzymes, and phase III transporters in rat liver and kidneys. For this purpose, SUN (25, 50 and 100 mg/kg) was injected IP into Wistar albino rats for 4 weeks; thereafter, the mRNA and protein expression levels of several XMEs and transporters were determined by RT-PCR and Western blot analysis, respectively. Real-time PCR analysis showed that SUN significantly induced the hepatic and renal CYP1A1, 1A2, 1B1, 2E1 and 4F4, whereas it inhibited CYP2C11 and 4A2. Furthermore, SUN specifically induced renal, but not hepatic, CYP2J3 and 3A2, while it induced only hepatic CYP4A1. With regard to phase II, SUN induced hepatic GSTA1 and UGT1A and renal NQO1 and UGT1A mRNA levels, whereas it inhibited renal GST1A expression. On the other hand, both renal and hepatic P-gp, MRP2 and BCRP transporters were significantly induced by SUN at the mRNA and protein expression levels. Importantly, these differential effects were associated with changes in oxidative stress genes and lipid peroxidation levels. In conclusion, SUN can serve as XME and transporters modulator, which potentially may counteract the efficacy of the treatment, adverse reactions and drug interactions in SUN treatment.

Molecular mechanisms of cardiotoxicity of gefitinib in vivo and in vitro rat cardiomyocyte: Role of apoptosis and oxidative stress.

Gefitinib (GEF) is a multi-targeted tyrosine kinase inhibitor with anti-cancer properties, yet few cases of cardiotoxicity has been reported as a significant side effect associated with GEF treatment. The main purpose of this study was to investigate the potential cardiotoxic effect of GEF and the possible mechanisms involved using in vivo and in vitro rat cardiomyocyte model. Treatment of rat cardiomyocyte H9c2 cell line with GEF (0, 1, 5, and 10µM) caused cardiomyocyte death and upregulation of hypertrophic gene markers, such as brain natriuretic peptides (BNP) and Beta-myosin heavy chain (ß-MHC) in a concentration-dependent manner at the mRNA and protein levels associated with an increase in the percentage of hypertrophied cardiac cells. Mechanistically, GEF treatment caused proportional and concentration-dependent increases in the mRNA and protein expression levels of apoptotic markers caspase-3 and p53 which was accompanied with marked increases in the percentage of H9c2 cells underwent apoptosis/necrosis as compared to control. In addition, oxidative stress marker (heme oxygenase-1, HO-1) and the formation of reactive oxygen species were increased in response to GEF treatment. At the in vivo level, treatment of Wistar albino rats for 21days with GEF (20 and 30mg/kg) significantly increased the cardiac enzymes (CK, CKmb, and LDH) levels associated with histopathological changes indicative of cardiotoxicity. Similarly, in vivo GEF treatment increased the mRNA and protein levels of BNP and ß-MHC whereas inhibited the antihypertrophoic gene (α-MHC) associated with increased the percentage of hypertrophied cells. Furthermore, the mRNA and protein expression levels of caspase-3, p53, and HO-1 genes and the percentage of apoptotic cells were significantly increased by GEF treatment, which was more pronounced at the 30mg/kg dose. In conclusion, GEF induces cardiotoxicity and cardiac hypertrophy in vivo and in vitro rat model through cardiac apoptotic cell death and oxidative stress pathways.

Effect of long-term human exposure to environmental heavy metals on the expression of detoxification and DNA repair genes.

The present study was designed to evaluate the influence of long-term environmental human exposure to three heavy metals, lead (Pb), cadmium (Cd), and mercury (Hg), on the expression of detoxifying, xenobiotic metabolizing, and DNA repair genes in Mahd Ad-Dahab city. The study groups consisted of 40 healthy male residents (heavy metal-exposed) and 20 healthy male from Riyadh city, 700 km away, and served as control group. The heavy metal-exposed group with high exposure to Pb, Cd, or Hg was divided into three subgroups Pb-, Cd-, and Hg-exposed groups, respectively. The mRNA expression levels of detoxifying, NQO1, HO-1, GSTA1, MT-1, and HSP70, were significantly decreased in all heavy metal-exposed group as compared to control group. This was accompanied with a proportional decrease in the expression of xenobiotic metabolizing gene, cytochrome P4501A1. On the other hand, the DNA repair gene OGG1 and the 8-OHdG level were dramatically inhibited in Cd-exposed group only.