Toxicity of cannabidiol and its metabolites in TM3 mouse Leydig cells: a comparison with primary human Leydig cells.

Cannabidiol (CBD), one of the major components extracted from the plant Cannabis sativa L., has been used as a prescription drug to treat seizures in many countries. CBD-induced male reproductive toxicity has been reported in animal models; however, the underlying mechanisms remain unclear. We previously reported that CBD induced apoptosis in primary human Leydig cells, which constitute the primary steroidogenic cell population in the testicular interstitium. In this study, we investigated the effects of CBD and its metabolites on TM3 mouse Leydig cells. CBD, at concentrations below 30 µM, reduced cell viability, induced G1 cell cycle arrest, and inhibited DNA synthesis. CBD induced apoptosis after exposure to high concentrations (≥ 50 µM) for 24 h or a low concentration (20 µM) for 6 days. 7-Hydroxy-CBD and 7-carboxy-CBD, the main CBD metabolites of CBD, exhibited the similar toxic effects as CBD. In addition, we conducted a time-course mRNA-sequencing analysis in both primary human Leydig cells and TM3 mouse Leydig cells to understand and compare the mechanisms underlying CBD-induced cytotoxicity. mRNA-sequencing analysis of CBD-treated human and mouse Leydig cells over a 5-day time-course indicated similar responses in both cell types. Mitochondria and lysosome dysfunction, oxidative stress, and autophagy were the major enriched pathways in both cell types. Taken together, these findings demonstrate comparable toxic effects and underlying mechanisms in CBD-treated mouse and primary human Leydig cells.

Evaluating the mode of action of perfluorooctanoic acid-induced liver tumors in male Sprague-Dawley rats using a toxicogenomic approach.

The mode of action (MOA) underlying perfluorooctanoic acid (PFOA)-induced liver tumors in rats is proposed to involve peroxisome proliferator-activated receptor α (PPARα) agonism. Despite clear PPARα activation evidence in rodent livers, the mechanisms driving cell growth remain elusive. Herein, we used dose-responsive apical endpoints and transcriptomic data to examine the proposed MOA. Male Sprague-Dawley rats were treated with 0, 1, 5, and 15 mg/kg PFOA for 7, 14, and 28 days via oral gavage. We showed PFOA induced hepatomegaly along with hepatocellular hypertrophy in rats. PPARα was activated in a dose-dependent manner. Toxicogenomic analysis revealed six early biomarkers (Cyp4a1, Nr1d1, Acot1, Acot2, Ehhadh, and Vnn1) in response to PPARα activation. A transient rise in hepatocellular DNA synthesis was demonstrated while Ki-67 labeling index showed no change. Transcriptomic analysis indicated no significant enrichment in pathways related to DNA synthesis, apoptosis, or the cell cycle. Key cyclins including Ccnd1, Ccnb1, Ccna2, and Ccne2 were dose-dependently suppressed by PFOA. Oxidative stress and the nuclear factor-κB signaling pathway were unaffected. Overall, evidence for PFOA-induced hepatocellular proliferation was transient within the studied timeframe. Our findings underscore the importance of considering inter-species differences and chemical-specific effects when evaluating the carcinogenic risk of PFOA in humans.

Use of Lentivirus-Based Method for Establishing TK6 Human Cell Lines Expressing Cytochrome P450 and its Applications in Genotoxicity Testing.

The human lymphoblastoid cell line TK6 stands out as the most widely employed human cell line in genotoxicity testing, as recommended by various testing guidelines for in vitro assessments. Nevertheless, like many testing cell lines, TK6 lacks functional phase I drug-metabolizing enzymes crucial for chemical genotoxicity evaluations. This protocol introduces a lentivirus-based methodology for establishing a panel of TK6-derived cell lines, each expressing one of 14 cytochrome P450s (CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, and CYP3A7). The utilization of a lentiviral expression system ensures stable transduction, offering notable advantages such as sustained transgene expression, high transduction efficiency, positive selection feasibility, and user-friendly application. Additionally, we present a detailed procedure for validating the enhanced expression of each CYP in the established cell lines through real-time PCR, western blotting, and mass spectrometry analysis. Lastly, we exemplify the application of these CYP-expressing TK6 cell lines in genotoxicity testing, employing a flow-cytometry-based in vitro micronucleus test. Published 2024. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Lentivirus production and transduction for TK6 cells Support Protocol: Selecting a single clone of CYP-expressing TK6 cells Basic Protocol 2: Validation of CYP expression in TK6 cell lines Basic Protocol 3: Application of transduced cell lines in flow-cytometry-based micronucleus assay.

Subacute Pulmonary Toxicity of Glutaraldehyde Aerosols in a Human In Vitro Airway Tissue Model.

Glutaraldehyde (GA) has been cleared by the Center for Devices and Radiological Health (CDRH) of the Food and Drug Administration (FDA) as a high-level disinfectant for disinfecting heat-sensitive medical equipment in hospitals and healthcare facilities. Inhalation exposure to GA is known to cause respiratory irritation and sensitization in animals and humans. To reproduce some of the known in vivo effects elicited by GA, we used a liquid aerosol exposure system and evaluated the tissue responses in a human in vitro airway epithelial tissue model. The cultures were treated at the air interface with various concentrations of GA aerosols on five consecutive days and changes in tissue function and structure were evaluated at select timepoints during the treatment phase and after a 7-day recovery period. Exposure to GA aerosols caused oxidative stress, inhibition of ciliary beating frequency, aberrant mucin production, and disturbance of cytokine and matrix metalloproteinase secretion, as well as morphological transformation. Some effects, such as those on goblet cells and ciliated cells, persisted following the 7-day recovery period. Of note, the functional and structural disturbances observed in GA-treated cultures resemble those found in ortho-phthaldehyde (OPA)-treated cultures. Furthermore, our in vitro findings on GA toxicity partially and qualitatively mimicked those reported in the animal and human survey studies. Taken together, observations from this study demonstrate that the human air-liquid-interface (ALI) airway tissue model, integrated with an in vitro exposure system that simulates human inhalation exposure, could be used for in vitro-based human hazard identification and the risk characterization of aerosolized chemicals.

Evaluating the Sub-Acute Toxicity of Formaldehyde Fumes in an In Vitro Human Airway Epithelial Tissue Model.

Formaldehyde (FA) is an irritating, highly reactive aldehyde that is widely regarded as an asthmagen. In addition to its use in industrial applications and being a product of combustion reaction and endogenous metabolism, FDA-regulated products may contain FA or release FA fumes that present toxicity risks for both patients and healthcare workers. Exposure to airborne FA is associated with nasal neoplastic lesions in both animals and humans. It is classified as a Group 1 carcinogen by International Agency for Research on Cancer (IARC) based on the increased incidence of cancer in animals and a known human carcinogen in the Report on Carcinogens by National Toxicology Program (NTP). Herein, we systematically evaluated the tissue responses to FA fumes in an in vitro human air-liquid-interface (ALI) airway tissue model. Cultures were exposed at the air interface to 7.5, 15, and 30 ppm of FA fumes 4 h per day for 5 consecutive days. Exposure to 30 ppm of FA induced sustained oxidative stress, along with functional changes in ciliated and goblet cells as well as possible squamous differentiation. Furthermore, secretion of the proinflammatory cytokines, IL-1ß, IL-2, IL-8, GM-CSF, TNF-a and IFN-γ, was induced by repeated exposures to FA fumes. Expression of MMP-1, MMP-3, MMP-7, MMP-10, MMP-12, and MMP-13 was downregulated at the end of the 5-day exposure. Although DNA-damage was not detected by the comet assay, FA exposures downregulated the DNA repair enzymes MGMT and FANCD2, suggesting its possible interference in the DNA repair capacity. Overall, a general concordance was observed between our in vitro responses to FA fume exposures and the reported in vivo toxicity of FA. Our findings provide further evidence supporting the application of the ALI airway system as a potential in vitro alternative for screening and evaluating the respiratory toxicity of inhaled substances.

In vitro effects of cannabidiol and its main metabolites in mouse and human Sertoli cells.

Cannabidiol (CBD) is a major cannabinoid present in extracts of the plant Cannabis sativa (marijuana). While the therapeutic effects of CBD on epilepsy have been demonstrated, less is understood regarding its potential adverse effects. Recent studies revealed that CBD induced toxicity in the male reproductive system of animal models. In this study, we used TM4, an immortalized mouse Sertoli cell line, and primary human Sertoli cells to evaluate the toxicities of CBD and its main metabolites, 7-carboxy-CBD and 7-hydroxy-CBD. CBD induced concentration- and time-dependent cytotoxicity in mouse and human Sertoli cells, which mainly resulted from the inhibition of the G1/S-phase cell cycle transition. CBD also inhibited DNA synthesis and downregulated key cell cycle proteins. Moreover, CBD reduced the mRNA and protein levels of a functional marker, Wilms' tumor 1. Similar to CBD, 7-carboxy-CBD and 7-hydroxy-CBD inhibited cellular proliferation and decreased DNA synthesis. 7-Carboxy-CBD was less cytotoxic than CBD, while 7-hydroxy-CBD showed comparable cytotoxicity to CBD in both mouse and human Sertoli cells. Compared to mouse Sertoli cells, CBD, 7-hydroxy-CBD, and 7-carboxy-CBD were more cytotoxic in human Sertoli cells. Our results indicate that CBD and its main metabolites can inhibit cell proliferation in mouse and human Sertoli cells.

Integration of transcriptome analysis with pathophysiological endpoints to evaluate cigarette smoke toxicity in an in vitro human airway tissue model.

Exposure to cigarette smoke (CS) is a known risk factor in the pathogenesis of smoking-caused diseases, such as chronic obstructive pulmonary diseases (COPD) and lung cancer. To assess the effects of CS on the function and phenotype of airway epithelial cells, we developed a novel repeated treatment protocol and comprehensively evaluated the progression of key molecular, functional, and structural abnormalities induced by CS in a human in vitro air-liquid-interface (ALI) airway tissue model. Cultures were exposed to CS (diluted with 0.5 L/min, 1.0 L/min, and 4.0 L/min clean air) generated from smoking five 3R4F University of Kentucky reference cigarettes under the International Organization for Standardization (ISO) machine smoking regimen, every other day for 4 weeks (3 days per week, 40 min/day). By integrating the transcriptomics-based approach with the in vitro pathophysiological measurements, we demonstrated CS-mediated effects on oxidative stress, pro-inflammatory cytokines and matrix metalloproteinases (MMPs), ciliary function, expression and secretion of mucins, and squamous cell differentiation that are highly consistent with abnormalities observed in airways of smokers. Enrichment analysis on the transcriptomic profiles of the ALI cultures revealed key molecular pathways, such as xenobiotic metabolism, oxidative stress, and inflammatory responses that were perturbed in response to CS exposure. These responses, in turn, may trigger aberrant tissue remodeling, eventually leading to the onset of respiratory diseases. Furthermore, changes of a panel of genes known to be disturbed in smokers with COPD were successfully reproduced in the ALI cultures exposed to CS. In summary, findings from this study suggest that such an integrative approach may be a useful tool for identifying genes and adverse cellular events caused by inhaled toxicants, like CS.

Transcriptome analysis reveals lung-specific miRNAs associated with impaired mucociliary clearance induced by cigarette smoke in an in vitro human airway tissue model.

Exposure to cigarette smoke (CS) is strongly associated with impaired mucociliary clearance (MCC), which has been implicated in the pathogenesis of CS-induced respiratory diseases, such as chronic obstructive pulmonary diseases (COPD). In this study, we aimed to identify microRNAs (miRNAs) that are associated with impaired MCC caused by CS in an in vitro human air-liquid-interface (ALI) airway tissue model. ALI cultures were exposed to CS (diluted with 0.5 L/min, 1.0 L/min, and 4.0 L/min of clean air) from smoking five 3R4F University of Kentucky reference cigarettes under the International Organization for Standardization (ISO) machine smoking regimen, every other day for 1 week (a total of 3 days, 40 min/day). Transcriptome analyses of ALI cultures exposed to the high concentration of CS identified 5090 differentially expressed genes and 551 differentially expressed miRNAs after the third exposure. Genes involved in ciliary function and ciliogenesis were significantly perturbed by repeated CS exposures, leading to changes in cilia beating frequency and ciliary protein expression. In particular, a time-dependent decrease in the expression of miR-449a, a conserved miRNA highly enriched in ciliated airway epithelia and implicated in motile ciliogenesis, was observed in CS-exposed cultures. Similar alterations in miR-449a have been reported in smokers with COPD. Network analysis further indicates that downregulation of miR-449a by CS may derepress cell-cycle proteins, which, in turn, interferes with ciliogenesis. Investigating the effects of CS on transcriptome profile in human ALI cultures may provide not only mechanistic insights, but potential early biomarkers for CS exposure and harm.

Characterization of cytochrome P450s (CYP)-overexpressing HepG2 cells for assessing drug and chemical-induced liver toxicity.

Hepatic metabolism catalyzed by the cytochrome P450 (CYP) superfamily affects liver toxicity associated with exposures to natural compounds and xenobiotic agents. Previously we generated a battery of HepG2-derived stable cell lines that individually express 14 CYPs (1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5, and 3A7). In this study, we comprehensively characterized each cell line for its CYP expression and enzyme activity. Specifically, we measured the mRNA expression, protein expression, and metabolite formation. Using CYP3A4, 2D6, and 2C9-overexpressing cells as representatives, we examined the stability of these cells in long-term cultures for up to 10 passages. The results showed that CYPs can be stably overexpressed for up to 10 cell culture passages without losing their activities. The robustness of responses to stimuli among the cells at different passages was also investigated in CYP3A4-overexpressing cells and the response to amiodarone and dronedarone showed no difference between the cells at the passage 2 and 10. Moreover, the mRNA expression level of most CYPs was higher in CYP-overexpressing HepG2 cells than that in HepaRG cells and primary human hepatocytes. This study confirmed the stability of CYP-overexpressing HepG2 cell lines and provided useful information for a broader use of these cells in pharmacologic and toxicologic research.

Toxicity of Ortho-phthalaldehyde Aerosols in a Human In Vitro Airway Tissue Model.

Ortho-phthalaldehyde (OPA) is a chemical disinfectant used for the high-level sterilization of heat-sensitive medical instruments. Although OPA is considered a safer alternative to glutaraldehyde, no exposure limits have been established for respiratory exposures to ensure the safety of OPA sterilization and the safe use of OPA-treated medical instruments. In order to address data gaps in the toxicological profile of OPA, we treated human in vitro air-liquid-interface (ALI) airway cultures at the air interface with various concentrations of OPA aerosols for 10 consecutive days. Temporal tissue responses were evaluated at multiple time points during the treatment phase as well as 10 days following the last exposure. The disturbance of glutathione (GSH) homeostasis occurred as early as 20 min following the first exposure, while oxidative stress persisted throughout the treatment phase, as indicated by the sustained induction of heme oxygenase-1 (HMOX-1) expression. Repeated exposures to OPA aerosols resulted in both functional and structural changes, including the inhibition of ciliary beating frequency, aberrant mucin production, decreases in airway secretory cells, and tissue morphological changes. While OPA-induced oxidative stress recovered to control levels after a 10 day recovery period, functional and structural alterations caused by the high concentration of OPA aerosols failed to fully recover over the observation period. These findings indicate that aerosolized OPA induces both transient and relatively persistent functional and structural abnormalities in ALI cultures under the conditions of the current study.

In vitro dosimetry analyses for acrolein exposure in normal human lung epithelial cells and human lung cancer cells.

Establishing accurate dosimetry is important for assessing the toxicity of xenobiotics as well as for comparing responses between different test systems. In this study, we used acrolein as a model toxicant and defined the concentration-response relationships of the key adverse responses in normal human bronchial epithelial (NHBE) cells and human mucoepidermoid pulmonary carcinoma (NCI-H292) cells. Direct trace analysis of intracellular free acrolein is extremely challenging, if not impossible. Therefore, we developed a new method for indirectly estimating the intracellular uptake of acrolein. A 10-min treatment was employed to capture the rapid occurrence of the key alkylation reactions of acrolein. Responses, including protein carbonylation, GSH depletion, and GSH-acrolein (GSH-ACR) adduct formation, were all linearly correlated with acrolein uptake in both cell types. Compared to the NCI-H292 mucoepidermoid carcinoma cells, NHBE cells were more sensitive to acrolein exposure. Furthermore, results from the time-course studies demonstrated that depletion and conjugation of GSH were the primary adverse events and directly associated with the cytotoxicity induced by acrolein. In summary, these data suggest that cell susceptibility to acrolein exposure is closely associated with acrolein uptake and formation of GSH-ACR adducts. The dosimetric analysis presented in this study may provide useful information for computational modeling and risk assessment of acrolein using different test systems.

The role of hepatic cytochrome P450s in the cytotoxicity of sertraline.

Sertraline, an antidepressant, is commonly used to manage mental health symptoms related to depression, anxiety disorders, and obsessive-compulsive disorder. The use of sertraline has been associated with rare but severe hepatotoxicity. Previous research demonstrated that mitochondrial dysfunction, apoptosis, and endoplasmic reticulum stress were involved in sertraline-associated cytotoxicity. In this study, we reported that after a 24-h treatment in HepG2 cells, sertraline caused cytotoxicity, suppressed topoisomerase I and IIα, and damaged DNA in a concentration-dependent manner. We also investigated the role of cytochrome P450 (CYP)-mediated metabolism in sertraline-induced toxicity using our previously established HepG2 cell lines individually expressing 14 CYPs (1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5, and 3A7). We demonstrated that CYP2D6, 2C19, 2B6, and 2C9 metabolize sertraline, and sertraline-induced cytotoxicity was significantly decreased in the cells expressing these CYPs. Western blot analysis demonstrated that the induction of É£H2A.X (a hallmark of DNA damage) and topoisomerase inhibition were partially reversed in CYP2D6-, 2C19-, 2B6-, and 2C9-overexpressing HepG2 cells. These data indicate that DNA damage and topoisomerase inhibition are involved in sertraline-induced cytotoxicity and that CYPs-mediated metabolism plays a role in decreasing the toxicity of sertraline.

Constitutive androstane receptor (CAR) mediates dieldrin-induced liver tumorigenesis in mouse.

Dieldrin has been shown to induce liver tumors selectively in mice. Although the exact mechanism is not fully understood, previous studies from our laboratory and others have shown that dieldrin induced liver tumors in mice through a non-genotoxic mechanism acting on tumor promotion stage. Two studies were performed to examine the role of nuclear receptor activation as a possible mode of action (MOA) for dieldrin-induced mouse liver tumors. In the initial study, male C57BL/6 mice (6- to 8-week old) were treated with dieldrin in diet (10 ppm) for 7, 14, and 28 days. Phenobarbital (PB), beta-naphthoflavone (BNF) and Di (2-ethylhexyl) phthalate (DEHP) were included as positive controls in this study for evaluating the involvement of CAR (constitutive androstane receptor), AhR (aryl hydrocarbon receptor) or PPARα (peroxisome proliferator activated receptor alpha) in the MOA of dieldrin hepatocarcinogenesis. A significant increase in hepatocyte DNA synthesis (BrdU incorporation) was seen in treated mice compared with the untreated controls. Analysis of the expression of the nuclear receptor responsive genes revealed that dieldrin induced a significant increase in the expression of genes specific to CAR activation (Cyp2b10, up to 400- to 2700-fold) and PXR activation (Cyp3a11, up to 5- to 11-fold) over untreated controls. The AhR target genes Cyp1a1 and Cyp1a2 were also slightly induced (2.0- to 3.7-fold and 1.7- to 2.8-fold, respectively). PPARα activation was not seen in the liver following dieldrin treatment. In addition, consistent with previous studies in our lab, treatment with dieldrin produced significant elevation in the hepatic oxidative stress. In a subsequent study using CAR, PXR, and CAR/PXR knockout mice, we confirmed that the dieldrin-induced liver effects in mouse were only mediated by the activation of CAR receptor. Based on these findings, we propose that dieldrin induced liver tumors in mice through a nuclear receptor CAR-mediated mode of action. The previously observed oxidative stress/damage may be an associated or modifying factor in the process of dieldrin-induced liver tumor formation subsequent to the CAR activation.

Performance of high-throughput CometChip assay using primary human hepatocytes: a comparison of DNA damage responses with in vitro human hepatoma cell lines.

Primary human hepatocytes (PHHs) are considered the "gold standard" for evaluating hepatic metabolism and toxicity of xenobiotics. In the present study, we evaluated the genotoxic potential of four indirect-acting (requiring metabolic activation) and six direct-acting genotoxic carcinogens, one aneugen, and five non-carcinogens that are negative or equivocal for genotoxicity in vivo in cryopreserved PHHs derived from three individual donors. DNA damage was determined over a wide range of concentrations using the CometChip technology and the resulting dose-responses were quantified using benchmark dose (BMD) modeling. Following a 24-h treatment, nine out of ten genotoxic carcinogens produced positive responses in PHHs, while negative responses were found for hydroquinone, aneugen colchicine and five non-carcinogens. Overall, PHHs demonstrated a higher sensitivity (90%) for detecting DNA damage from genotoxic carcinogens than the sensitivities previously reported for HepG2 (60%) and HepaRG (70%) cells. Quantitative analysis revealed that most of the compounds produced comparable BMD10 values among the three types of hepatocytes, while PHHs and HepaRG cells produced similar BMD1SD values. Evidence of sex- and ethnicity-related interindividual variation in DNA damage responses was also observed in the PHHs. A literature search for in vivo Comet assay data conducted in rodent liver tissues demonstrated consistent positive/negative calls for the compounds tested between in vitro PHHs and in vivo animal models. These results demonstrate that CometChip technology can be applied using PHHs for human risk assessment and that PHHs had higher sensitivity than HepaRG cells for detecting genotoxic carcinogens in the CometChip assay.

Development and Application of TK6-derived Cells Expressing Human Cytochrome P450s for Genotoxicity Testing.

Metabolism plays a key role in chemical genotoxicity; however, most mammalian cells used for in vitro genotoxicity testing lack effective metabolizing enzymes. We recently developed a battery of TK6-derived cell lines that individually overexpress 1 of 8 cytochrome P450s (CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C9, 2C19, and 3A4) using a lentiviral expression system. The increased expression and metabolic function of each individual CYP in each established cell line were confirmed using real-time PCR, Western blotting, and mass spectrometry analysis; the parental TK6 cells and empty vector (EV) transduced cells had negligible CYP levels. Subsequently, we evaluated these cell lines using 2 prototypical polyaromatic hydrocarbon mutagens, 7,12-dimethylbenz[a]anthracene (DMBA) and benzo[a]pyrene (B[a]P), that require metabolic activation to exert their genotoxicity. DMBA-induced cytotoxicity, phosphorylation of histone H2A.X, and micronucleus formation were significantly increased in TK6 cells with CYP1A1, 1B1, 2B6, and 2C19 expression as compared with EV controls. B[a]P significantly increased cytotoxicity, DNA damage, and chromosomal damage in TK6 cells overexpressing CYP1A1 and 1B1 when compared with EV controls. B[a]P also induced micronucleus formation in TK6 cells expressing CYP1A2. These results suggest that our CYP-expressing TK6 cell system can be used to detect the genotoxicity of compounds requiring metabolic transformation.

Apoptosis contributes to the cytotoxicity induced by amodiaquine and its major metabolite N-desethylamodiaquine in hepatic cells.

Amodiaquine (ADQ), an antimalarial drug used in endemic areas, has been reported to be associated with liver toxicity; however, the mechanism underlying its hepatoxicity remains unclear. In this study, we examined the cytotoxicity of ADQ and its major metabolite N-desethylamodiaquine (NADQ) and the effect of cytochrome P450 (CYP)-mediated metabolism on ADQ-induced cytotoxicity. After a 48-h treatment, ADQ and NADQ caused cytotoxicity and induced apoptosis in HepG2 cells; NADQ was slightly more toxic than ADQ. ADQ treatment decreased the levels of anti-apoptotic Bcl-2 family proteins, which was accompanied by an increase in the levels of pro-apoptotic Bcl-2 family proteins, indicating that ADQ-induced apoptosis was mediated by the Bcl-2 family. NADQ treatment markedly increased the phosphorylation of JNK, extracellular signal-regulated kinase (ERK1/2), and p38, indicating that NADQ-induced apoptosis was mediated by MAPK signaling pathways. Metabolic studies using microsomes obtained from HepG2 cell lines overexpressing human CYPs demonstrated that CYP1A1, 2C8, and 3A4 were the major enzymes that metabolized ADQ to NADQ and that CYP1A2, 1B1, 2C19, and 3A5 also metabolized ADQ, but to a lesser extent. The cytotoxicity of ADQ was increased in CYP2C8 and 3A4 overexpressing HepG2 cells compared to HepG2/CYP vector cells, confirming that NADQ was more toxic than ADQ. Moreover, treatment of CYP2C8 and 3A4 overexpressing HepG2 cells with ADQ increased the phosphorylation of JNK, ERK1/2, and p38, but not the expression of Bcl-2 family proteins. Our findings indicate that ADQ and its major metabolite NADQ induce apoptosis, which is mediated by members of the Bcl-2 family and the activation of MAPK signaling pathways, respectively.

Assessing the respiratory toxicity of dihydroxyacetone using an in vitro human airway epithelial tissue model.

Dihydroxyacetone (DHA) is an approved color additive used in sunless tanning lotions. Recently, there has been an increased use of DHA in sunless tanning booths in a manner that could result in its inhalation during application. In the present study, we have evaluated the potential for DHA causing toxicity via inhalation using a human air-liquid-interface (ALI) in vitro airway epithelial tissue model. ALI airway models have a close structural and functional resemblance to the in vivo airway epithelium, and thus data generated in these models may have relevance for predicting human responses. To simulate in vivo exposure conditions, we employed a method for liquid aerosol generation that mimics the physical form of inhaled chemicals and used doses of DHA and an exposure frequency reflecting human respiratory exposures during tanning sessions. Compared to the vehicle control, cilia beating frequency (CBF) and MUC5AC secretion were significantly decreased after each exposure. However, time-course studies indicated that both CBF and MUC5AC secretion returned to normal levels within 3 days after the treatment. Matrix metalloproteinase (MMP) release, on the other hand, was decreased 24 h after the first exposure and its level returned to baseline after 5 exposures. No significant morphological changes occurred in the DHA-treated cultures after 5 weekly exposures. Our findings indicate that DHA, at concentrations likely to be experienced by humans, has transient toxic effects on human airway ALI cultures.

Quantitative comparison of in vitro genotoxicity between metabolically competent HepaRG cells and HepG2 cells using the high-throughput high-content CometChip assay.

In vitro genotoxicity testing that employs metabolically active human cells may be better suited for evaluating human in vivo genotoxicity than current bacterial or non-metabolically active mammalian cell systems. In the current study, 28 compounds, known to have different genotoxicity and carcinogenicity modes of action (MoAs), were evaluated over a wide range of concentrations for the ability to induce DNA damage in human HepG2 and HepaRG cells. DNA damage dose-responses in both cell lines were quantified using a combination of high-throughput high-content (HTHC) CometChip technology and benchmark dose (BMD) quantitative approaches. Assays of metabolic activity indicated that differentiated HepaRG cells had much higher levels of cytochromes P450 activity than did HepG2 cells. DNA damage was observed for four and two out of five indirect-acting genotoxic carcinogens in HepaRG and HepG2 cells, respectively. Four out of seven direct-acting carcinogens were positive in both cell lines, with two of the three negatives being genotoxic mainly through aneugenicity. The four chemicals positive in both cell lines generated HTHC Comet data in HepaRG and HepG2 cells with comparable BMD values. All the non-genotoxic compounds, including six non-genotoxic carcinogens, were negative in HepaRG cells; five genotoxic non-carcinogens also were negative. Our results indicate that the HTHC CometChip assay detects a greater proportion of genotoxic carcinogens requiring metabolic activation (i.e., indirect carcinogens) when conducted with HepaRG cells than with HepG2 cells. In addition, BMD genotoxicity potency estimate is useful for quantitatively evaluating CometChip assay data in a scientifically rigorous manner.

Effects of cellular differentiation in human primary bronchial epithelial cells: Metabolism of 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone.

Many of the toxicants in tobacco smoke undergo biotransformation in the lungs of smokers, both to reactive and to detoxified derivatives. Human air-liquid-interface (ALI) airway tissue models have emerged as an advanced in vitro model for evaluating the toxicity of inhaled substances; however, the metabolic potential of these cultures has not been evaluated extensively. In this study, we compared the metabolic activities of an ALI tissue model to the undifferentiated normal human primary bronchial epithelial (NHBE) cells from which it was derived. Measurement of the basal levels of gene expression for 84 phase I drug metabolism enzymes indicated that most genes were upregulated in ALI cultures compared to NHBE cells. Furthermore, the enzymatic activities of three cytochrome P450s involved in the bioactivation of tobacco-specific nitrosamines were higher in the ALI cultures, and the bioactivation of 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK), as measured by the formation of two of its major metabolites, i.e., keto acid and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), was significantly greater in the ALI cultures. Finally, NNK was a direct-acting genotoxicant in the ALI cultures, while the genotoxicity of NNK was detected in NHBE cells only in the presence of an exogenous liver S9 activation system. Taken together, our findings demonstrate the greater metabolic potential of well-differentiated ALI cultures than primary NHBE cells, supporting the potential use of ALI airway cultures as an alternative in vitro model for evaluating inhaled toxicants that require metabolic transformation.