Pharmacokinetic Analysis of Nicotine and Its Metabolites (Cotinine and trans-3'-Hydroxycotinine) in Male Sprague-Dawley Rats Following Nose-Only Inhalation, Oral Gavage, and Intravenous Infusion of Nicotine.

Nicotine is an alkaloid found in tobacco. Human exposure to nicotine primarily occurs through the use of tobacco products. To date, limited nicotine pharmacokinetic data in animals have been reported. This study exposed male Sprague-Dawley rats to vehicle (and/or air) or four doses of nicotine via nose-only inhalation (INH), oral gavage (PO), and intravenous (IV) infusion. Plasma, six tissues (brain, heart, lung, liver, kidney, and muscle), and urine were collected at multiple timepoints from 5 minutes to 48 hours post-dose. The concentrations of nicotine, cotinine, and trans-3'-hydroxycotinine (3-OH-cotinine) were determined, and the pharmacokinetic profiles were compared among the four doses for each route. The results indicated that after single nicotine dose, nicotine bioavailability was 53% via PO. Across all the administration routes and doses, nicotine was quickly distributed to all six tissues; kidney had the highest nicotine and cotinine levels, and the lung had the highest 3-OH-cotinine levels; nicotine was metabolized extensively to cotinine and cotinine was metabolized to a lesser extent to 3-OH-cotinine; the elimination of plasma nicotine, cotinine, and 3-OH-cotinine followed first-order kinetics; plasma nicotine had a shorter half-life than cotinine or 3-OH-cotinine; the half-lives of plasma nicotine, cotinine, and 3-OH-cotinine were dose- and route-independent; and nicotine and cotinine were major urinary excretions followed by 3-OH-cotinine. Nicotine, cotinine, and 3-OH-cotinine levels in plasma, tissues, and urine exhibited dose-dependent increases. These study findings improve our understanding of the pharmacokinetics of nicotine, cotinine, and 3-OH-cotinine across different routes of exposure.

Botanical-induced toxicity: Liver injury and botanical-drug interactions. A report on a society of Toxicology Annual Meeting symposium.

Botanical supplements and herbal products are widely used by consumers for various purported health benefits, and their popularity is increasing. Some of these natural products can have adverse effects on liver function and/or interact with prescription and over-the-counter (OTC) medications. Ensuring the safety of these readily available products is a crucial public health concern; however, not all regulatory authorities require premarket safety review and/or testing. To address and discuss these and other emerging needs related to botanical safety, a symposium was held at the Society of Toxicology Annual Meeting in Salt Lake City (UT) on March 11, 2024. The symposium addressed the latest research on botanical-induced liver toxicity and botanical-drug interactions, including new approach methods to screen for toxicity, challenges in assessing the safety of botanicals, and relating human adverse events to specific products. The presentations and robust panel discussion between the speakers and audience highlighted the need for further research and collaboration to improve the safety of botanical supplements and herbal products, with the ultimate goal of protecting consumer health. Although utility of many of the modern tools presented in the symposium requires further study, the synergistic efforts of diverse experts hold promise for effective prediction and evaluation of botanical-induced hepatotoxicity and botanical-drug interaction potential.

90-day nose-only inhalation toxicity study of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in Sprague-Dawley rats.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the key tobacco-specific nitrosamines that plays an important role in human lung carcinogenesis. Repeated dose inhalation toxicity data on NNK, particularly relevant to cigarette smoking, however, is surprisingly limited. Hence, there is a lack of direct information available on the carcinogenic and potential non-carcinogenic effects of NNK via inhalational route exposure. In the present study, the subchronic inhalation toxicity of NNK was evaluated in Sprague Dawley rats. Both sexes (9-10 weeks age; 23 rats/sex/group) were exposed by nose-only inhalation to air, vehicle control (75% propylene glycol), or 0.2, 0.8, 3.2, or 7.8 mg/kg body weight (BW)/day of NNK (NNK aerosol concentrations: 0, 0, 0.0066, 0.026, 0.11, or 0.26 mg/L air) for 1 h/day for 90 consecutive days. Toxicity was evaluated by assessing body weights; food consumption; clinical pathology; histopathology; organ weights; blood, urine, and tissue levels of NNK, its major metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and their glucuronides (reported as total NNK, tNNK, and total NNAL, tNNAL, respectively); tissue levels of the DNA adduct O6-methylguanine; blood and bone marrow micronucleus (MN) frequency; and bone marrow DNA strand breaks (comet assay). The results showed that NNK exposure caused multiple significant adverse effects, with the most sensitive endpoint being non-neoplastic lesions in the nose. Although the genotoxic biomarker O6-methylguanine was detected, genotoxicity from NNK exposure was negative in the MN and comet assays. The Lowest-Observed-Adverse-Effect-Level (LOAEL) was 0.8 mg/kg BW/day or 0.026 mg/L air of NNK for 1 h/day for both sexes. The No-Observed-Adverse-Effect-Level (NOAEL) was 0.2 mg/kg BW/day or 0.0066 mg/L air of NNK for 1 h/day for both sexes. The results of this study provide new information relevant to assessing the human exposure hazard of NNK.

14-Day Nose-Only Inhalation Toxicity and Haber's Rule Study of NNK in Sprague-Dawley Rats.

4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the key tobacco-specific nitrosamines that plays an important role in human lung carcinogenesis. However, repeated inhalation toxicity data on NNK, which is more directly relevant to cigarette smoking, are currently limited. In the present study, the subacute inhalation toxicity of NNK was evaluated in Sprague Dawley rats. Both sexes (9-10 weeks age; 16 rats/sex/group) were exposed by nose-only inhalation to air, vehicle control (75% propylene glycol), or 0.8, 3.2, 12.5, or 50 mg/kg body weight (BW)/day of NNK (NNK aerosol concentrations: 0, 0, 0.03, 0.11, 0.41, or 1.65 mg/L air) for 1 h/day for 14 consecutive days. Toxicity was evaluated by assessing body and organ weights; food consumption; clinical pathology; histopathology observations; blood, urine, and tissue levels of NNK, its major metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and their glucuronides (reported as total NNK, tNNK, and total NNAL, tNNAL, respectively); O6-methylguanine DNA adduct formation; and blood and bone marrow micronucleus frequency. Whether the subacute inhalation toxicity of NNK followed Haber's Rule was also determined using additional animals exposed 4 h/day. The results showed that NNK exposure caused multiple significant adverse effects, with the most sensitive endpoint being non-neoplastic histopathological lesions in the nose. The lowest-observed-adverse-effect level (LOAEL) was 0.8 mg/kg BW/day or 0.03 mg/L air for 1 h/day for both sexes. An assessment of Haber's Rule indicated that 14-day inhalation exposure to the same dose at a lower concentration of NNK aerosol for a longer time (4 h daily) resulted in greater adverse effects than exposure to a higher concentration of NNK aerosol for a shorter time (1 h daily).

Toxicokinetic and Genotoxicity Study of NNK in Male Sprague Dawley Rats Following Nose-Only Inhalation Exposure, Intraperitoneal Injection, and Oral Gavage.

The tobacco-specific nitrosamine NNK [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone] is found in tobacco products and tobacco smoke. NNK is a potent genotoxin and human lung carcinogen; however, there are limited inhalation data for the toxicokinetics (TK) and genotoxicity of NNK in vivo. In the present study, a single dose of 5 × 10-5, 5 × 10-3, 0.1, or 50 mg/kg body weight (BW) of NNK, 75% propylene glycol (vehicle control), or air (sham control) was administered to male Sprague-Dawley (SD) rats (9-10 weeks age) via nose-only inhalation (INH) exposure for 1 h. For comparison, the same doses of NNK were administered to male SD rats via intraperitoneal injection (IP) and oral gavage (PO). Plasma, urine, and tissue specimens were collected at designated time points and analyzed for levels of NNK and its major metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and tissue levels of DNA adduct O6-methylguanine by LC/MS/MS. TK data analysis was performed using a non-linear regression program. For the genotoxicity subgroup, tissues were collected at 3 h post-dosing for comet assay analysis. Overall, the TK data indicated that NNK was rapidly absorbed and metabolized extensively to NNAL after NNK administration via the three routes. The IP route had the greatest systemic exposure to NNK. NNK metabolism to NNAL appeared to be more efficient via INH than IP or PO. NNK induced significant increases in DNA damage in multiple tissues via the three routes. The results of this study provide new information and understanding of the TK and genotoxicity of NNK.

Nonanimal toxicology testing approaches for traditional and deemed tobacco products in a complex regulatory environment: Limitations, possibilities, and future directions.

The evaluation of tobacco products is complex due to a multitude of factors including product diversity, limited testing standards, and variability in user behavior. Alternative approaches in current testing paradigms have limitations that generally truncate their applicability beyond screening for hazard identification; this is also true for toxicological evaluations of tobacco products. In a regulatory context, results from tobacco product toxicity assessments are extrapolated to the in vivo condition to assess human health relevance at the individual and population level. A key limitation of alternative approaches is the difficulty and uncertainty in extrapolating results to adverse outcomes relevant to chronic tobacco exposures in humans. This difficulty and uncertainty are increased when comparing toxicological outcomes between tobacco products. Given that the interpretation and quantification of differences in assay results (e.g., mutagenicity) for tobacco product comparison may be inconclusive, the predictive value of these approaches for human risk of relevant downstream pathologies (e.g., carcinogenesis) can be limited. Development and validation of fit-for-purpose alternative approaches that are predictive of human toxicity and dose response assays with adequate sensitivity and specificity for product comparisons would help advance the field of predictive toxicology.

Proposed Mode of Action for Acrolein Respiratory Toxicity Associated with Inhaled Tobacco Smoke.

This article presents a mode of action (MOA) analysis that identifies key mechanisms in the respiratory toxicity of inhaled acrolein and proposes key acrolein-related toxic events resulting from the inhalation of tobacco smoke. Smoking causes chronic obstructive pulmonary disorder (COPD) and acrolein has been previously linked to the majority of smoking-induced noncancer respiratory toxicity. In contrast to previous MOA analyses for acrolein, this MOA focuses on the toxicity of acrolein in the lower respiratory system, reflecting the exposure that smokers experience upon tobacco smoke inhalation. The key mechanisms of acrolein toxicity identified in this proposed MOA include (1) acrolein chemical reactivity with proteins and other macromolecules of cells lining the respiratory tract, (2) cellular oxidative stress, including compromise of the important anti-oxidant glutathione, (3) chronic inflammation, (4) necrotic cell death leading to a feedback loop where necrosis-induced inflammation leads to more necrosis and oxidative damage and vice versa, (5) tissue remodeling and destruction, and (6) loss of lung elasticity and enlarged lung airspaces. From these mechanisms, the proposed MOA analysis identifies the key cellular processes in acrolein respiratory toxicity that consistently occur with the development of COPD: inflammation and necrosis in the middle and lower regions of the respiratory tract. Moreover, the acrolein exposures that occur as a result of smoking are well above exposures that induce both inflammation and necrosis in laboratory animals, highlighting the importance of the role of acrolein in smoking-related respiratory disease.