Sex and reproductive development impact skin mucosal epithelium immunity, antimicrobial capacity, and up-regulation of immune-related gene of goldfish (Carassius auratus).

This study evaluated the epidermis mucosal capacity of goldfish (Carassius auratus) during different stages of reproductive development in both females and males. In this regard, the activity of mucolytic immune enzymes, i.e., lysozyme, complement and peroxidase, as well as the activity of alkaline phosphatase (ALP) were evaluated. There were five stages for females i.e., immature (f1), cortical alveoli (f2), early and late-vitellogenesis (vtg) (f3 and f4) and ripe (f5); as well as two stages for males spermatogenesis (m1) and spermiation (m2). Some stages were also examined for the mucosal antimicrobial activity against specific pathogens. The results showed that the mucosal lysozyme activity increased significantly during vitellogenesis (P < 0.05), but no lysozyme activity was detected in plasma. On the contrary, the complement activity was only observed in female plasma, and it was significantly higher at f3 compared to the other developmental stages. Both the plasma and mucosal ALP and peroxidase activities showed a significant increase by female reproductive development with the highest amounts at f4. Contrary to the female, no significant changes were observed in plasma and mucosal immune agents and biochemistry of the male. The f5-staged goldfish showed the highest antimicrobial activities against Gram-positive bacteria, i.e., Streptococcus faecium, Staphylococcus aureus and Micrococcus luteus (P < 0.05). Our results also represented the up-regulation of lysozyme (c-lys) gene expression by effects of female maturational development in ovary, liver and skin, while male goldfish showed no significant changes in c-lys expression. Moreover, there were positive correlations between c-lys expression, mucosal lysozyme activity and calcium levels in females (P < 0.01). Overall, our findings revealed that vtg process improves mucosal innate immunity that leads to activate antimicrobial components at spawning season.

Mucus interaction to improve gastrointestinal retention and pharmacokinetics of orally administered nano-drug delivery systems.

Oral delivery of therapeutics is the preferred route of administration due to ease of administration which is associated with greater patient medication adherence. One major barrier to oral delivery and intestinal absorption is rapid clearance of the drug and the drug delivery system from the gastrointestinal (GI) tract. To address this issue, researchers have investigated using GI mucus to help maximize the pharmacokinetics of the therapeutic; while mucus can act as a barrier to effective oral delivery, it can also be used as an anchoring mechanism to improve intestinal residence. Nano-drug delivery systems that use materials which can interact with the mucus layers in the GI tract can enable longer residence time, improving the efficacy of oral drug delivery. This review examines the properties and function of mucus in the GI tract, as well as diseases that alter mucus. Three broad classes of mucus-interacting systems are discussed: mucoadhesive, mucus-penetrating, and mucolytic drug delivery systems. For each class of system, the basis for mucus interaction is presented, and examples of materials that inform the development of these systems are discussed and reviewed. Finally, a list of FDA-approved mucoadhesive, mucus-penetrating, and mucolytic drug delivery systems is reviewed. In summary, this review highlights the progress made in developing mucus-interacting systems, both at a research-scale and commercial-scale level, and describes the theoretical basis for each type of system.

Intestinal mucus-derived metabolites modulate virulence of a clade 8 enterohemorrhagic Escherichia coli O157:H7.

The human colonic mucus is mainly composed of mucins, which are highly glycosylated proteins. The normal commensal colonic microbiota has mucolytic activity and is capable of releasing the monosaccharides contained in mucins, which can then be used as carbon sources by pathogens such as Enterohemorrhagic Escherichia coli (EHEC). EHEC can regulate the expression of some of its virulence factors through environmental sensing of mucus-derived sugars, but its implications regarding its main virulence factor, Shiga toxin type 2 (Stx2), among others, remain unknown. In the present work, we have studied the effects of five of the most abundant mucolytic activity-derived sugars, Fucose (L-Fucose), Galactose (D-Galactose), N-Gal (N-acetyl-galactosamine), NANA (N-Acetyl-Neuraminic Acid) and NAG (N-Acetyl-D-Glucosamine) on EHEC growth, adhesion to epithelial colonic cells (HCT-8), and Stx2 production and translocation across a polarized HCT-8 monolayer. We found that bacterial growth was maximum when using NAG and NANA compared to Galactose, Fucose or N-Gal, and that EHEC adhesion was inhibited regardless of the metabolite used. On the other hand, Stx2 production was enhanced when using NAG and inhibited with the rest of the metabolites, whilst Stx2 translocation was only enhanced when using NANA, and this increase occurred only through the transcellular route. Overall, this study provides insights on the influence of the commensal microbiota on the pathogenicity of E. coli O157:H7, helping to identify favorable intestinal environments for the development of severe disease.

Mucolytic bacteria license pathobionts to acquire host-derived nutrients during dietary nutrient restriction.

Pathobionts employ unique metabolic adaptation mechanisms to maximize their growth in disease conditions. Adherent-invasive Escherichia coli (AIEC), a pathobiont enriched in the gut mucosa of patients with inflammatory bowel disease (IBD), utilizes diet-derived L-serine to adapt to the inflamed gut. Therefore, the restriction of dietary L-serine starves AIEC and limits its fitness advantage. Here, we find that AIEC can overcome this nutrient limitation by switching the nutrient source from the diet to the host cells in the presence of mucolytic bacteria. During diet-derived L-serine restriction, the mucolytic symbiont Akkermansia muciniphila promotes the encroachment of AIEC to the epithelial niche by degrading the mucus layer. In the epithelial niche, AIEC acquires L-serine from the colonic epithelium and thus proliferates. Our work suggests that the indirect metabolic network between pathobionts and commensal symbionts enables pathobionts to overcome nutritional restriction and thrive in the gut.

Spray-dried multidrug particles for pulmonary co-delivery of antibiotics with N-acetylcysteine and curcumin-loaded PLGA-nanoparticles.

Nowadays, the resistance of bacterial biofilms towards the available antibiotics is a severe problem. Therefore, many efforts were devoted to develop new formulations using nanotechnology. We have developed an inhalable microparticle formulation using spray-drying combining multiple drugs: an antibiotic (tobramycin, ciprofloxacin or azithromycin), N-acetylcysteine (NAC), and curcumin (Cur). The use of PLGA nanoparticles (NP) also allowed incorporating curcumin to facilitate spray drying and modify the release of some compounds. The aerosolizable microparticles formulations were characterized in terms of size, morphology, and aerodynamic properties. Biocompatibility when tested on macrophage-like cells was acceptable after 20 h exposure for concentrations up to at least 32 µg/mL. Antibacterial activity of free drugs versus drugs in the multiple drug formulations was evaluated on P. aeruginosa in the same range. When co-delivered the efficacy of tobramycin was enhanced compared to the free drug for the 1 µg/mL concentration. The combinations of azithromycin and ciprofloxacin with NAC and Cur did not show an improved antibacterial activity. Bacteria-triggered cytokine release was not inhibited by free antibiotics, except for TNF-α. In contrast, the application of NAC and the addition of curcumin-loaded PLGA NPs showed a higher potential to inhibit TNF-α, IL-8, and IL-1ß release. Overall, the approach described here allows simultaneous delivery of antibacterial, mucolytic, and anti-inflammatory compounds in a single inhalable formulation and may therefore pave the way for a more efficient therapy of pulmonary infections.

Mucus permeating self-emulsifying drug delivery systems (SEDDS): About the impact of mucolytic enzymes.

This study aimed to improve the mucus permeating properties of self-emulsifying drug delivery systems (SEDDS) by anchoring lipidized bromelain, papain and trypsin using palmitoyl chloride. SEDDS containing enzyme-palmitate conjugates were characterized regarding droplet size and zeta potential. Their mucus permeating properties were evaluated by Transwell diffusion and rotating tube method using fluorescein diacetate (FDA) as marker. Degree of substitution of modified enzymes was 35.3%, 47.8% and 38.5% for bromelain-palmitate, papain-palmitate and trypsin-palmitate, respectively. SEDDS as control and SEDDS containing enzyme-palmitate conjugates displayed a droplet size less than 50nm and 180-312nm as well as a zeta potential of -3 to -4 and -4 to -5mV, respectively. The highest percentage of permeation was achieved by introducing 5% papain-palmitate into SEDDS. It could enhance the mucus permeation of SEDDS in porcine intestinal mucus 4.6-fold and 2-fold as evaluated by Transwell diffusion and rotating tube method, respectively. It is concluded that mucus permeation of SEDDS can be strongly improved by incorporation of enzyme-palmitate conjugates.

[The application of N-acetylcysteine in optimization of specific pharmacological therapies]. / Zastosowanie N-acetylocysteiny do optymalizacji specyficznych terapii farmakologicznych.

Based on the analysis of data from clinical trials it could be postulated that N-acetylcysteine has a positive impact on the treatment of various diseases. However, less is known about specific molecular and physiological mechanisms underlying the reported therapeutic effects. N-acetylcysteine (NAC, N-acetyl-L-cysteine) is an amino acid derivative containing a thiol group. It is a precursor of L-cysteine and glutathione. NAC is well absorbed and safe for the body at doses up to 300 mg per kg of body weight. Side effects are relatively rare. NAC is used as an mucolytic agent in adjunctive therapy of respiratory diseases causing the retention of secretions, as well as an antidote in the treatment of paracetamol poisoning. Moreover, NAC protects against the toxic effects of reactive oxygen species and their active metabolites. NAC is involved in free radical scavenging processes via several independent mechanisms, including a direct reduction of free radicals, providing substrates for oxidation-reduction reactions and activation of antioxidant enzymes. In the blood, NAC decreases the level of low density lipoprotein peroxidation. In various tissues, NAC may increase the levels of glutathione and cysteine and stimulate the superoxide dismutase action. NAC is used as a supplement in the treatment of various diseases associated with impaired exterior and intracellular oxidative balance. NAC increases the concentrations of amino acids and their derivatives, including cysteine, cystine, and glutathione. It also stabilizes the antioxidant status of the cells and the intercellular spaces. NAC changes the levels of transcription factors, modifying the transcription of selected genes and acting on the protein translation. It works on the activation of several enzymes in the cells and outside the cells. Based on the analysis of data from clinical trials it can be concluded, that an administration of NAC may be beneficial for these groups of patients, in whom the reversible accumulation and the negative action of free radicals was observed.

Nanocomplexes for gene therapy of respiratory diseases: Targeting and overcoming the mucus barrier.

Gene therapy, i.e. the delivery and expression of therapeutic genes, holds great promise for congenital and acquired respiratory diseases. Non-viral vectors are less toxic and immunogenic than viral vectors, although they are characterized by lower efficiency. However, they have to overcome many barriers, including inflammatory and immune mediators and cells. The respiratory and airway epithelial cells, the main target of these vectors, are coated with a layer of mucus, which hampers the effective reaching of gene therapy vectors carrying either plasmid DNA or small interfering RNA. This barrier is thicker in many lung diseases, such as cystic fibrosis. This review summarizes the most important advancements in the field of non-viral vectors that have been achieved with the use of nanoparticulate (NP) systems, composed either of polymers or lipids, in the lung gene delivery. In particular, different strategies of targeting of respiratory and airway lung cells will be described. Then, we will focus on the two approaches that attempt to overcome the mucus barrier: coating of the nanoparticulate system with poly(ethylene glycol) and treatment with mucolytics. Our conclusions are: 1) Ligand and physical targeting can direct therapeutic gene expression in specific cell types in the respiratory tract; 2) Mucopenetrating NPs are endowed with promising features to be useful in treating respiratory diseases and should be now advanced in pre-clinical trials. Finally, we discuss the development of such polymer- and lipid-based NPs in the context of in vitro and in vivo disease models, such as lung cancer, as well as in clinical trials.

S-carboxymethyl-L-cysteine and it (R/S)-S-oxides in beagle dog plasma and hepatic cytosol.

1. Incubation of beagle hepatic cytosol, under conditions promoting phenylalanine hydroxylase activity, led to the formation of the sulfoxide derivatives of S-carboxymethyl-L-cysteine, N-acetyl-S-carboxymethyl-L-cysteine, S-methyl-L-cysteine and N-acetyl-S-methyl-L-cysteine. Thiodiglycolic acid was not a substrate. Enzyme kinetic parameters (Km, Vmax) were derived indicating S-carboxymethyl-L-cysteine had the greatest clearance; no enantioselective preference was observed for this S-oxygenation reaction. 2. Following oral administration of S-carboxymethyl-L-cysteine to beagle dogs, the parent substance and its sulfoxide were the only compounds identified in the plasma. Pharmacokinetic data have been obtained indicating that the small amount of sulfoxide formed persisted within the body for longer than the parent material, but that the majority of the ingested dose remained in the administered sulfide form. 3. The sulfide moiety within the muco-regulatory drug, S-carboxymethyl-L-cysteine, is thought to be vital as it acts as a free radical scavenger, resulting in the inactive sulfoxide. Additional extensive enyzme-mediated sulfoxidation would decrease the amount of active sulfide available. In the dog this appears to not be an issue, signalling possible exploitation for therapeutic benefit in treating airway disease.

Identifying the elusive sites of tyrosyl radicals in cytochrome c peroxidase: implications for oxidation of substrates bound at a site remote from the heme.

The location of the Trp radical and the catalytic function of the [Fe(IV)═O Trp191(•+)] intermediate in cytochrome c peroxidase (CcP) are well-established; however, the unambiguous identification of the site(s) for the formation of tyrosyl radical(s) and their possible biological roles remain elusive. We have now performed a systematic investigation of the location and reactivity of the Tyr radical(s) using multifrequency Electron Paramagnetic Resonance (EPR) spectroscopy combined with multiple-site Trp/Tyr mutations in CcP. Two tyrosines, Tyr71 and Tyr236, were identified as those contributing primarily to the EPR spectrum of the tyrosyl radical, recorded at 9 and 285 GHz. The EPR characterization also showed that the heme distal-side Trp51 is involved in the intramolecular electron transfer between Tyr71 and the heme and that formation of Tyr71(•) and Tyr236(•) is independent of the [Fe(IV)═O Trp191(•+)] intermediate. Tyr71 is located in an optimal position to mediate the oxidation of substrates binding at a site, more than 20 Šfrom the heme, which has been reported recently in the crystal structures of CcP with bound guaicol and phenol [Murphy, E. J., et al. (2012) FEBS J. 279, 1632-1639]. The possibility of discriminating the radical intermediates by their EPR spectra allowed us to identify Tyr71(•) as the reactive species with the guaiacol substrate. Our assignment of the surface-exposed Tyr236 as the other radical site agrees well with previous studies based on MNP labeling and protein cross-linking [Tsaprailis, G., and English, A. M. (2003) JBIC, J. Biol. Inorg. Chem. 8, 248-255] and on its covalent modification upon reaction of W191G CcP with 2-aminotriazole [Musah, R. A., and Goodin, D. B. (1997) Biochemistry 36, 11665-11674]. Accordingly, while Tyr71 acts as a true reactive intermediate for the oxidation of certain small substrates that bind at a site remote from the heme, the surface-exposed Tyr236 would be more likely related to oxidative stress signaling, as previously proposed. Our findings reinforce the view that CcP is the monofunctional peroxidase that most closely resembles its ancestor enzymes, the catalase-peroxidases, in terms of the higher complexity of the peroxidase reaction [Colin, J., et al. (2009) J. Am. Chem. Soc. 131, 8557-8563]. The strategy used to identify the elusive Tyr radical sites in CcP may be applied to other heme enzymes containing a large number of Tyr and Trp residues and for which Tyr (or Trp) radicals have been proposed to be involved in their peroxidase or peroxidase-like reaction.

La hoja de hiedra en el tratamiento de las afecciones de vías respiratorias: evidencias preclínicas y clínicas / Ivy leaf in the treatment of respiratory conditions: preclinical and clinical evidences

La hoja de hiedra es una droga vegetal empleada desde la antigüedad con fines medicinales, cuyos extractos son ampliamente utilizados en el tratamiento de afecciones de vías respiratorias debido, principalmente, a su actividad expectorante y espasmolítica. Los principios activos son saponósidos triterpénicos pentacíclicos, siendo el mayoritario el hederacósido C, heterósido bidesmosídico que en el organismo se transforma en el mododesmósido alpha-hederina, principal responsable de la actividad farmacológica. Su mecanismo de acción, recientemente descrito, se relaciona con la inhibición de la internalización de receptores B2-adrenérgicos, lo cual determina una mayor reactividad de los mismos dando lugar a una fluidificación de las secreciones bronquiales, favoreciendo su expectoración. Los estudios clínicos evidencian tanto la eficacia del extracto hidroetanólico (EtOH 30%, RDE_: 5-7,5:1) en el tratamiento de infecciones agudas del tracto respiratorio acompañadas de tos y de enfermedades bronquiales inflamatorias crónicas, como su excelente tolerabilidad en niños y adultos (AU)

Ivy leaf is an herbal drug used since ancient times for medicinal purposes. Its extracts are widely used in the treatment of respiratory conditions, mainly due to its expectorant and spasmolytic activities. The active principles are pentacyclic y triterpene saponins, the major one being hederacoside C, a bidesmosidic glycoside that in the body is transformed into the monodesmosidic alpha-hederin, which is the main responsible of the pharmacological activity. Its mechanism of action, recently described, is related to the inhibition of the internalisation of B2-adrenergic receptors, which determines their higher reactivity resulting in an increase of secretolysis and bronchodilation. Clinical studies demonstrate both the efficacy of the hydroalcoholic extract (EtOH 30%, DER:5-7,5:1) in the treatment of acute respiratory tract infections accompanied by cough and chronic inflammatory bronchial conditions, as well as its excellent tolerability in children and adults (AU)

Membrane permeability in the gastrointestinal tract: the interplay between microclimate pH and transporters.

Some examples of pH- and transporter-dependent permeability, determined in side-by-side diffusion cells, are summarized. We investigated the polarized transport in the mucosal-to-serosal direction of monocarboxylic acid-type drugs through the excised rat jejunal tissue and an artificial membrane. We established that, in vitro, these substances are most probably not transported by monocarboxylate transporter 1, but by passive pH-dependent transport. We also studied various influences on the permeability of fluorescein, a low permeability marker, through isolated rat intestinal segments, Caco-2 cell monolayers, and an artificial membrane. Polarized transport of fluorescein in the serosal-to-mucosal direction through the rat jejunum by multidrug resistance-associated protein was triggered by the addition of D-glucose to the mucosal side, while the pH-dependent increase of fluorescein influx is presumably the consequence of a monocarboxylate transporter and a member of the organic-anion transporting polypeptide family. With permeability experiments through the excised segments of rat small intestine, we ascertained that ciprofloxacin is a low-permeability drug and has higher and pH-dependent transport in the mucosal-to-serosal direction than in the opposite direction. We also established that neither the permeability of fluoroquinolones nor their solubility in different buffers was influenced by the interactions with metal cations.

Simultaneous detection of N-acetyl-L-cysteine and physiological low molecular mass thiols in plasma by capillary electrophoresis.

N-acetyl-L-cysteine (NAC) is a therapeutic drug widely used as mucolytic agent in the treatment of respiratory diseases. Recently it has been proposed that NAC administration may modify the plasma levels of low molecular weight thiols (LMW) like cysteine, homocysteine and glutathione, though it has been still debated if their plasma concentration increases or decreases during the therapy. Therefore research calls for methods able to analyze simultaneously NAC and the other plasma LMW thiols in order to evaluate if NAC is able to modify plasma thiols concentration and in particular to reduce homocysteine levels in hyperhomocysteinemia. In this paper we present a new capillary electrophoresis method that allows a baseline separation of plasma NAC from the physiological thiols. The proposed method has been utilized to measure the drug and the physiological LMW thiols in NAC administered chronic obstructive broncho-pneumopathy (COPB) disease patients.

Measurement of phenylalanine monooxygenase (PAH) activities.

Mammalian phenylalanine monooxygenase (phenylalaninase, phenylalanine hydroxylase, PAH; EC 1.14.16.1) is a member of the large aromatic amino acid hydrolase cohort of enzymes that include tyrosine monooxygenase and tryptophane monooxygenase. PAH is a non-heme-iron-dependent protein that normally catalyzes the C-oxidation of phenylalanine (Phe) to tyrosine (Tyr) in the presence of BH(4), utilizing molecular dioxygen as an additional substrate. However, over recent years, the presumed narrow substrate specificity of PAH has been questioned and catalytic activity towards alternative xenobiotic substrates (both environmental and drugs) has been reported. Like the cytochrome P450 system, PAH is able to oxidize both aliphatic and aromatic carbon centers in addition to undertaking the S-oxidation of aliphatic thioethers (including the two mucoactive drugs S-carboxymethyl-L-cysteine and S-methyl-L-cysteine).

Effect of COPD treatments on MRP1-mediated transport in bronchial epithelial cells.

BACKGROUND: Smoking is the principle risk factor for development of chronic obstructive pulmonary disease (COPD). Multidrug resistance-associated protein 1 (MRP1) is known to protect against toxic compounds and oxidative stress, and might play a role in protection against smoke-induced disease progression. We questioned whether MRP1-mediated transport is influenced by pulmonary drugs that are commonly prescribed in COPD. METHODS: The immortalized human bronchial epithelial cell line 16HBE14o- was used to analyze direct in vitro effects of budesonide, formoterol, ipratropium bromide and N-acetylcysteine (NAC) on MRP1-mediated transport. Carboxyfluorescein (CF) was used as a model MRP1 substrate and was measured with functional flow cytometry. RESULTS: Formoterol had a minor effect, whereas budesonide concentration-dependently decreased CF transport by MRP1. Remarkably, addition of formoterol to the highest concentration of budesonide increased CF transport. Ipratropium bromide inhibited CF transport at low concentrations and tended to increase CF transport at higher levels. NAC increased CF transport by MRP1 in a concentration-dependent manner. CONCLUSIONS: Our data suggest that, besides their positive effects on respiratory symptoms, budesonide, formoterol, ipratropium bromide, and NAC modulate MRP1 activity in bronchial epithelial cells. Further studies are required to assess whether stimulation of MRP1 activity is beneficial for long-term treatment of COPD.

Protective effects of erdosteine and vitamins C and E combination on ischemia-reperfusion-induced lung oxidative stress and plasma copper and zinc levels in a rat hind limb model.

The aim of this study was to investigate the protective effects of erdosteine and vitamins C and E (VCE) on the lungs after performing hind limb ischemia-reperfusion (I/R) by assessing oxidative stress, plasma copper (Cu), and zinc (Zn) analysis. The animals were divided randomly into four groups as nine rats each as follows: control, I/R, I/R plus erdosteine, and I/R plus VCE combination. I/R period for 60 min was performed on the both hind limbs of all the rats in the groups of I/R, erdosteine with I/R, VCE with I/R allowing 120 min of reperfusion. The animals received orally erdosteine one time in a day and 3 days before I/R in the erdosteine group. In the VCE group, the animals VCE combination received one time in a day and 3 days before I/R, although placebo was given to control and I/R group animals. Lung lipid peroxidation (malondialdehyde [MDA]) level, superoxide dismutase (SOD), and catalase activities were increased, although lung glutathione (GSH) and plasma Zn levels decreased in I/R group in lung tissue compared with the control group. Serum MDA level, creatine kinase, and lactate dehydrogenase activities were increased in I/R group compared with the control. Lung MDA and plasma Zn levels and lung SOD activity were decreased by erdosteine administration, whereas lung GSH levels after I/R increased. The plasma Zn levels and lung SOD activity were decreased by VCE administration, although the plasma Cu and lung GSH levels increased after I/R. In conclusion, erdosteine has an antioxidant role on the values in the rat model, and it has more protective affect than in VCE in attenuating I/R-induced lung injury in rats.

In vitro metabolism of isoline, a pyrrolizidine alkaloid from Ligularia duciformis, by rodent liver microsomal esterase and enhanced hepatotoxicity by esterase inhibitors.

Isoline, a major retronecine-type pyrrolizidine alkaloid (PA) from the Chinese medicinal herb Ligularia duciformis, was suggested to be the most toxic known PA. Its in vitro metabolism was thus examined in rat and mouse liver microsomes, and its toxicity was compared with that of clivorine and monocrotaline after i.p. injection in mice. Isoline was more rapidly metabolized by both microsomes than clivorine and monocrotaline and converted to two polar metabolites M1 and M2, which were spectroscopically determined to be bisline (a deacetylated metabolite of isoline) and bisline lactone, respectively. Both metabolites were formed in the presence or absence of an NADPH-generating system with liver microsomes but not cytosol. Their formation was completely inhibited by the esterase inhibitors, triorthocresyl phosphate (TOCP) and phenylmethylsulfonyl fluoride, but not at all or partially by cytochrome P450 (P450) inhibitors, alpha-naphthoflavone and proadifen (SKF 525A), respectively. These results demonstrated that both metabolites were produced by microsomal esterase(s) but not P450 isozymes. The esterase(s) involved showed not only quite different activities but also responses to different inhibitors in rat and mouse liver microsomes, suggesting that different key isozyme(s) or combinations might be responsible for the deacetylation of isoline. Isoline injected i.p. into mice induced liver-specific toxicity that was much greater than that with either clivorine or monocrotaline, as judged by histopathology as well as serum alanine aminotransferase and aspartate aminotransferase levels. Isoline-induced hepatotoxicity was remarkably enhanced by the esterase inhibitor TOCP but was reduced by the P450 inhibitor SKF 525A, indicating that rodent hepatic esterase(s) played a principal role in the detoxification of isoline via rapid deacetylation in vivo.