Exploring Neurobehaviour in Zebrafish Embryos as a Screening Model for Addictiveness of Substances

Tobacco use is the leading cause of preventable death worldwide and is highly addictive. Nicotine is the main addictive compound in tobacco, but less is known about other components and additives that may contribute to tobacco addiction. The zebrafish embryo (ZFE) has been shown to be a good model to study the toxic effects of chemicals on the neurological system and thus may be a promising model to study behavioral markers of nicotine effects, which may be predictive for addictiveness. We aimed to develop a testing protocol to study nicotine tolerance in ZFE using a locomotion test with light-dark transitions as behavioral trigger. Behavioral experiments were conducted using three exposure paradigms: (1) Acute exposure to determine nicotine’s effect and potency. (2) Pre-treatment with nicotine dose range followed by a single dose of nicotine, to determine which pre-treatment dose is sufficient to affect the potency of acute nicotine. (3) Pre-treatment with a single dose combined with acute exposure to a dose range to confirm the hypothesized decreased potency of the acute nicotine exposure. These exposure paradigms showed that (1) acute nicotine exposure decreased ZFE activity in response to dark conditions in a dose-dependent fashion; (2) pre-treatment with increasing concentrations dose-dependently reversed the effect of acute nicotine exposure; and (3) a fixed pre-treatment dose of nicotine induced a decreased potency of the acute nicotine exposure. This effect supported the induction of tolerance to nicotine by the pre-treatment, likely through neuroadaptation. The interpretation of these effects, particularly in view of prediction of dependence and addictiveness, and suitability of the ZFE model to test for such effects of other compounds than nicotine, are discussed.

Type VI secretion system facilitates fitness, homeostasis, and competitive advantages for environmental adaptability and efficient nicotine biodegradation

Gram-negative bacteria employ secretion systems to translocate proteinaceous effectors from the cytoplasm to the extracellular milieu, thus interacting with the surrounding environment or micro-niche. It is known that bacteria can benefit from type VI secretion system (T6SS) by transporting ions to combat reactive oxygen species (ROS). Here, we report that T6SS activities conferred bacterial tolerance to nicotine-induced oxidative stress in Pseudomonas sp. strain JY-Q, a highly active nicotine degradation strain isolated from tobacco waste extract. AA098_13375 was identified to encode a dual-functional effector with anti-microbial and anti-ROS activities. Wild type strain JY-Q grew better than AA098_13375 deletion mutant in nicotine-containing medium by antagonizing increased intracellular ROS levels. It was, therefore, tentatively designated as TseN (Type VI Secretion system Effector for Nicotine tolerance), of which homologs were observed to be broadly ubiquitous in Pseudomonas species. TseN was identified as a Tse6-like bacteriostatic toxin via monitoring intracellular NAD+. TseN presented potential antagonism against ROS to fine tune the heavy traffic of nicotine metabolism in strain JY-Q. It is feasible that the dynamic tuning of NAD+ driven by TseN could satisfy demands from nicotine degradation with less cytotoxicity. In this scenario, T6SS involves a fascinating accommodation cascade that prompts constitutive biotransformation of N-heterocyclic aromatics by improving bacterial robustness/growth. In summary, T6SS in JY-Q mediated resistance to oxidative stress and promoted bacterial fitness via a contact-independent growth competitive advantage besides the well-studied T6SS-dependent antimicrobial activities. IMPORTANCE Mixtures of various pollutants and co-existence of numerous species of organisms are usually found in the adverse environments. Scientific community concerning biodegradation of nitrogen-heterocyclic contaminants commonly focused on screening functional enzymes transforming pollutants into intermediates of attenuated toxicity or for primary metabolism. Here we identified dual roles of T6SS effector TseN in Pseudomonas sp. JY-Q capable of degrading nicotine. T6SS could deliver TseN to kill competitors, and provide growth advantage by contact-independent pattern. TseN could monitor intracellular NAD+ level by its hydrolase activity, conferring cytotoxicity on competitive rivals but metabolic homeostasis on JY-Q. Moreover, JY-Q could be protected from TseN toxicity by its immunity protein TsiN. In conclusion, we found that TseN with cytotoxicity to bacterial competitors facilitated nicotine tolerance of JY-Q. We therefore revealed a working model between T6SS and nicotine metabolism. This finding indicates that multiple diversified weapons have been evolved by bacteria for their growth and robustness.

Characterization of UDP-Glucuronosyltransferases and the Potential Contribution to Nicotine Tolerance in Myzus persicae

Uridine diphosphate (UDP)-glycosyltransferases (UGTs) are major phase II detoxification enzymes involved in glycosylation of lipophilic endobiotics and xenobiotics, including phytoalexins. Nicotine, one of the most abundant secondary plant metabolites in tobacco, is highly toxic to herbivorous insects. Plant-herbivore competition is the major impetus for the evolution of large superfamilies of UGTs and other detoxification enzymes. However, UGT functions in green peach aphid (Myzus persicae) adaptation are unknown. In this study, we show that UGT inhibitors (sulfinpyrazone and 5-nitrouracil) significantly increased nicotine toxicity in M. persicae nicotianae, suggesting that UGTs may be involved in nicotine tolerance. In total, 101 UGT transcripts identified in the M. persicae genome/transcriptome were renamed according to the UGT Nomenclature Committee guidelines and grouped into 11 families, UGT329, UGT330, UGT339, UGT341–UGT345, and UGT348–UGT350, with UGT344 containing the most (57). Ten UGTs (UGT330A3, UGT339A2, UGT341A6, UGT342B3, UGT343C3, UGT344D5, UGT344D8, UGT348A3, UGT349A3, and UGT350A3) were highly expressed in M. persicae nicotianae compared to M. persicae sensu stricto. Knockdown of four UGTs (UGT330A3, UGT344D5, UGT348A3, and UGT349A3) significantly increased M. persicae nicotianae sensitivity to nicotine, suggesting that UGT expression in this subspecies may be associated with nicotine tolerance and thus host adaptation. This study reveals possible UGTs relevant to nicotine adaptation in tobacco-consuming M. persicae nicotianae, and the findings will facilitate further validation of the roles of these UGTs in nicotine tolerance.

Characterization of UDP-Glucuronosyltransferases and the Potential Contribution in Nicotine Tolerance in Myzus persicae

Uridine diphosphate (UDP)-glycosyltransferases (UGTs) are major phase II detoxification enzymes involved in glycosylation of lipophilic endobiotics and xenobiotics, including phytoalexins. Nicotine, one of the most abundant secondary plant metabolites, is highly toxic to herbivorous insects. Plant-herbivore competition has been the major impetus for evolution of large superfamilies of UGTs and other detoxification enzymes. However, UGT functions in Myzus persicae adaptation have remained unknown. In this study, UGT inhibitors (sulfinpyrazone and 5-nitrouracil) significantly increased nicotine toxicity in M. persicae nicotianae, suggesting UGTs may be involved in nicotine tolerance. One hundred one UGT transcripts identified from the M. persicae genome/transcriptome were renamed according to UGT Nomenclature Committee guidelines and clustered into 11 families: UGT329, UGT330, UGT339, UGT341-UGT345, and UGT348-UGT350. UGT344 contained the most UGTs (fifty-seven). Ten UGTs (UGT330A3, UGT339A2, UGT341A6, UGT342B3, UGT343C3, UGT344D5, UGT344D8, UGT348A3, UGT349A3 and UGT350A3) were overexpressed in M. persicae nicotianae compared with M. persicae sensu stricto. UGT330A3/UGT344D5/UGT348A3/UGT349A3 knockdown significantly increased M. persicae nicotianae nicotine sensitivity, suggesting UGT upregulation in this subspecies may be associated with nicotine tolerance and thus host adaptation. This study reveals possible UGTs relevant to nicotine adaptation in tobacco-consuming M. persicae nicotianae and might facilitate further validation of the roles of these UGTs in nicotine tolerance.

Unexpected Loss of Sensitivity to the nAChR Antagonist Activity of Mecamylamine and DHβE in Nicotine-Tolerant C57BL/6J Mice

There has always been interest in developing nAChR antagonists as smoking cessation aids, to add to nAChR agonists (e.g., nicotine replacement) already used for that indication. Previous studies have demonstrated that daily nicotine treatment confers tolerance to some of the effects of nicotine, as well as cross-tolerance to other nAChR agonists. The current study assessed the extent to which antagonism of nicotine varies as a function of daily nicotine treatment. The rate-decreasing and hypothermic effects of nicotine, as well as antagonism of those effects, were examined in C57BL/6J mice before, during treatment with, and after discontinuation of three daily injections of 1.78 mg/kg nicotine. The nonselective nAChR antagonist mecamylamine and the β2 nAChR antagonist DHβE were studied in combination with nicotine. The ED50 values of nicotine to produce rate-decreasing and hypothermic effects were, respectively, 0.44 and 0.82 mg/kg prior, 1.6 and 3.2 mg/kg during, and 0.74 and 1.1 mg/kg after discontinuation of daily nicotine treatment. Prior to daily nicotine treatment, mecamylamine decreased response rate and rectal temperature; however, during daily nicotine, mecamylamine (up to 5.6 mg/kg) only decreased rectal temperature. DHβE (up to 5.6 mg/kg) when studied prior to daily nicotine decreased rectal temperature, but that decrease was abolished during chronic nicotine treatment. Mecamylamine and DHβE antagonized the rate-decreasing and hypothermic effects of nicotine before and after daily nicotine; however, during daily nicotine, mecamylamine and DHβE antagonized only the hypothermic effects of nicotine. The differential antagonism of rate-decreasing and hypothermic effects implicates differential involvement of nAChR subtypes. The decreased capacity of mecamylamine and DHβE to antagonize nicotine during chronic nicotine treatment may indicate that their effectiveness as smoking cessations might vary as a function of nicotine tolerance and dependence.

Nicotine administration and withdrawal affect survival in systemic inflammation models

How different regimens of nicotine administration and withdrawal affect systemic inflammation is largely unknown. We studied the effects of chronic and acute nicotine administration and of nicotine withdrawal on the outcome of aseptic and septic systemic inflammation. Male C57BL/6 mice were implanted with subcutaneous osmotic pumps (to deliver nicotine) and intrabrain telemetry probes (to measure temperature). Aseptic inflammation was induced by lipopolysaccharide (40 mg/kg ip); sepsis was induced by cecal ligation and puncture. The chronic nicotine administration group received nicotine (28 mg·kg−1·day−1) for 2 wk before the induction of inflammation and continued receiving nicotine until the end of the experiment; the acute nicotine administration group received saline for 2 wk and nicotine thereafter; the nicotine withdrawal group received nicotine for 2 wk and saline thereafter; and the no-nicotine group was infused with saline throughout the experiment. Compared with no nicotine, the chronic nicotine administration did not affect survival in either model of inflammation, possibly due to the development of nicotine tolerance. The acute nicotine administration increased the survival rate in aseptic inflammation from 11 to 33% (possibly by suppressing inflammation) but worsened the outcome of sepsis (possibly because the suppression of inflammation promoted microbial proliferation). Oppositely to acute nicotine, nicotine withdrawal increased the survival rate in sepsis from 18 to 40%. The effects on survival were not due to changes in body temperature. We conclude that acute nicotine administration and nicotine withdrawal affect survival in systemic inflammation and that these effects strongly depend on whether inflammation is aseptic or septic.