"Pharm-ecology" of diet shifting: biotransformation of plant secondary compounds in creosote (Larrea tridentata) by a woodrat herbivore, Neotoma lepida

Diet switching in mammalian herbivores may necessitate a change in the biotransformation enzymes used to process plant secondary compounds (PSCs). We investigated differences in the biotransformation system in the mammalian herbivore, Neotoma lepida, after a radical shift in diet and secondary compound composition. Populations of N. lepida in the Mojave Desert have evolved over the past 10,000 years to feed on creosote (Larrea tridentata) from an ancestral state of consuming juniper (Juniperus osteosperma). This dietary shift represents a marked change in the dietary composition of PSCs in that creosote leaves are coated with phenolic resin, whereas juniper is high in terpenes but lacks phenolic resin. We quantified the enzyme activity of five major groups of biotransformation enzymes (cytochrome P450s, NAD(P)H:quinone oxidoreductase, glutathione conjugation, sulfation, and glucuronidation) recognized for their importance to mammalian biotransformation for the elimination of foreign compounds. Enzyme activities were compared between populations of Mojave and Great Basin woodrats fed control and creosote diets. In response to creosote, the Mojave population had greater levels of cytochrome P450s (CYP2B, CYP1A) and glutathione conjugation liver enzymes compared with the Great Basin population. Our results suggest that elevated levels of cytochrome P450s and glutathione conjugation enzymes in the Mojave population may be the underlying biotransformation mechanisms that facilitate feeding on creosote.     

Expression of biotransformation genes in woodrat (Neotoma) herbivores on novel and ancestral diets: identification of candidate genes responsible for dietary shifts

The ability of herbivores to switch diets is thought to be governed by biotransformation enzymes. To identify potential biotransformation enzymes, we conducted a large-scale study on the expression of biotransformation enzymes in herbivorous woodrats ( Neotoma lepida ). We compared gene expression in a woodrat population from the Great Basin that feeds on the ancestral diet of juniper to one from the Mojave Desert that putatively switched from feeding on juniper to feeding on creosote. Juniper and creosote have notable differences in secondary chemistry, and thus, should require different biotransformation enzymes for detoxification. Individuals from each population were fed juniper and creosote diets separately. After the feeding trials, hepatic mRNA was extracted and hybridized to laboratory rat microarrays. Hybridization of woodrat samples to biotransformation probes on the array was 87%, resulting in a total of 224 biotransformation genes that met quality control standards. Overall, we found large differences in expression of biotransformation genes when woodrats were fed juniper vs. creosote. Mojave woodrats had greater expression of 10× as many biotransformation genes as did Great Basin woodrats on a creosote diet. We identified 24 candidate genes that may be critical in the biotransformation of creosote toxins. Superoxide dismutase, a free radical scavenger, was also expressed to a greater extent by the Mojave woodrats and may be important in controlling oxidative damage during biotransformation. The results are consistent with the hypothesis that biotransformation enzymes limit diet switching and that woodrats in the Mojave have evolved a unique strategy for the biotransformation of creosote toxins.     

Xenobiotic metabolism of plant secondary compounds in juniper (Juniperus monosperma) by specialist and generalist woodrat herbivores, genus Neotoma

Mammalian herbivores routinely consume diets laden with often-toxic xenobiotics, yet the manner in which mammalian herbivores detoxify these plant secondary compounds (PSC) is largely unknown. Theory predicts that specialists rely more heavily on functionalization pathways whereas generalists rely on conjugation pathways to metabolize PSC in their diet. We took a pharmacological approach to determine how a specialist (Neotoma stephensi) of juniper foliage (Juniperus monosperma) and a generalist (N. albigula) may process the same dietary PSC. We investigated the xenobiotic metabolizing enzymes of the specialist and generalist on a control diet and a low (25%) juniper diet. We also examined enzyme activities in the specialist on a high (70%) juniper diet. We assayed for cytochrome P450 concentration and biotransformation activities of three specific cytochrome P450 isozymes (CYP1A, CYP2B, CYP3A), NAD(P)H:quinone oxidoreductase, glutathione conjugation, sulfation and glucuronidation. Results provide partial evidence for the hypothesis in that the specialist and generalist consuming juniper at a level similar to their natural diet, differ in the level of conjugation enzyme activity with generalists having higher activity overall than specialists.     

Cell-specific expression of CYP2A5 in the mouse respiratory tract: effects of olfactory toxicants.

We performed a detailed analysis of mouse cytochrome P450 2A5 (CYP2A5) expression by in situ hybridization (ISH) and immunohistochemistry (IHC) in the respiratory tissues of mice. The CYP2A5 mRNA and the corresponding protein co-localized at most sites and were predominantly detected in the olfactory region, with an expression in sustentacular cells, Bowman's gland, and duct cells. In the respiratory and transitional epithelium there was no or only weak expression. The nasolacrimal duct and the excretory ducts of nasal and salivary glands displayed expression, whereas no expression occurred in the acini. There was decreasing expression along the epithelial linings of the trachea and lower respiratory tract, whereas no expression occurred in the alveoli. The hepatic CYP2A5 inducers pyrazole and phenobarbital neither changed the CYP2A5 expression pattern nor damaged the olfactory mucosa. In contrast, the olfactory toxicants dichlobenil and methimazole induced characteristic changes. The damaged Bowman's glands displayed no expression, whereas the damaged epithelium expressed the enzyme. The CYP2A5 expression pattern is in accordance with previously reported localization of protein and DNA adducts and the toxicity of some CYP2A5 substrates. This suggests that CYP2A5 is an important determinant for the susceptibility of the nasal and respiratory epithelia to protoxicants and procarcinogens.     

Cytochrome P450 2B Diversity and Dietary Novelty in the Herbivorous, Desert Woodrat (Neotoma lepida)

Detoxification enzymes play a key role in plant-herbivore interactions, contributing to the on-going evolution of ecosystem functional diversity. Mammalian detoxification systems have been well studied by the medical and pharmacological industries to understand human drug metabolism; however, little is known of the mechanisms employed by wild herbivores to metabolize toxic plant secondary compounds. Using a wild rodent herbivore, the desert woodrat (Neotoma lepida), we investigated genomic structural variation, sequence variability, and expression patterns in a multigene subfamily involved in xenobiotic metabolism, cytochrome P450 2B (CYP2B). We hypothesized that differences in CYP2B expression and sequence diversity could explain differential abilities of woodrat populations to consume native plant toxins. Woodrats from two distinct populations were fed diets supplemented with either juniper (Juniperus osteosperma) or creosote bush (Larrea tridentata), plants consumed by woodrats in their respective desert habitats. We used Southern blot and quantitative PCR to determine that the genomic copy number of CYP2B in both populations was equivalent, and similar in number to known rodent copy number. We compared CYP2B expression patterns and sequence diversity using cloned hepatic CYP2B cDNA. The resulting sequences were very diverse, and clustered into four major clades by amino acid similarity. Sequences from the experimental treatments were distributed non-randomly across a CYP2B tree, indicating unique expression patterns from woodrats on different diets and from different habitats. Furthermore, within each major CYP2B clade, sequences shared a unique combination of amino acid residues at 13 sites throughout the protein known to be important for CYP2B enzyme function, implying differences in the function of each major CYP2B variant. This work is the most comprehensive investigation of the genetic diversity of a detoxification enzyme subfamily in a wild mammalian herbivore, and contributes an initial genetic framework to our understanding of how a wild herbivore responds to critical changes in its diet.     

Testing the diet-breadth trade-off hypothesis: differential regulation of novel plant secondary compounds by a specialist and a generalist herbivore

Specialist herbivores are predicted to have evolved biotransformation pathways that can process large doses of secondary compounds from the plant species on which they specialize. It is hypothesized that this physiological specialization results in a trade-off such that specialists may be limited in ability to ingest novel plant secondary compounds (PSCs). In contrast, the generalist foraging strategy requires that herbivores alternate consumption of plant species and PSC types to reduce the possibility of over-ingestion of any particular PSC. The ability to behaviorally regulate is a key component of this strategy. These ideas underpin the prediction that in the face of novel PSCs, generalists should be better able to maintain body mass and avoid toxic consequences compared to specialists. We explored these predictions by comparing the feeding behavior of two herbivorous rodents: a juniper specialist, Neotoma stephensi, and a generalist, Neotoma albigula, fed diets with increasing concentrations of phenolic resin extracted from the creosote bush (Larrea tridentata), which produces a suite of PSCs novel to both species. The specialist lost more mass than the generalist during the 15-day trial. In addition, although the specialist and generalist both regulated phenolic resin intake by reducing meal size while on the highest resin concentration (4%), the generalist began to regulate intake on the 2% diet. The ability of the generalist to regulate intake at a lower PSC concentration may be the source of the generalist’s performance advantage over the specialist. These data provide evidence for the hypothesis that the specialist’s foraging strategy may result in behavioral as well as physiological trade-offs in the ability to consume novel PSCs.     

Characterization of biotransformation enzyme activities in primary rat proximal tubular cells

The proximal tubule is a frequent target for nephrotoxic compounds due to it's ability to transport and accumulate xenobiotics and their metabolites, as well as by the presence of an organ-selective set of biotransformation enzymes. The aim of the present study was to characterize the activities of different biotransformation enzymes during primary culturing of rat proximal tubular cells (PT cells). Specific marker substrates for determining cytochrome P450 (CYP450) activity of primary cultured PT cells include 7-ethoxyresorufin (CYP1A1), caffeine (CYP1A), testosterone (CY2B/C, CYP3A), tolbutamide (CYP2C) and dextromethorphan (CYP2D1). Activities of the CYP450 isoenzymes decreased considerably during culture with the greatest loss in activity within 24 h of culture. In addition, expression of CYP450 apoprotein, including CYP1A, CYP2C, CYP2D, CYP2E and CYP4A, was detected in microsomes from freshly isolated PT cells by immunoblotting using specific antibodies. CYP2B and CYP3A apoprotein could not be detected. Activity of the phase II biotransformation enzymes GST, GGT, beta-lyase and UGT was determined with 1-chloro-2,4-dinitrobenzene, L-glutamic acid gamma-(7-amido-4-methyl-coumarin), S-(1,1,2,2-tetrafluoroethyl)-L-cysteine and 1-naphthol, respectively, as marker substrates. Activity of the phase II enzymes remained more stable and, in contrast to CYP450 activity, significant activity was still expressed after 1 week of PT cell culture. Thus, despite the obvious advantages of PT cells as an in-vitro model for studies of biotransformation mediated toxicity, the strong time dependency of especially phase I and, to a lesser extent, phase II biotransformation activities confers limitations to their application.     

Differential hepatic gene expression of a dietary specialist (Neotoma stephensi) and generalist (Neotoma albigula) in response to juniper (Juniperus monosperma) ingestion

Dietary specialization is thought to be rare in mammalian herbivores because of limitations of their detoxification system in processing large doses of a single type of plant secondary compound (PSC). Therefore, in order to specialize on a single species of plant, mammalian herbivores must have a highly efficient detoxification system for the particular types of PSCs they ingest. Using microarray technology, we looked at the expression of hepatic genes of a dietary specialist, Neotoma stephensi, and a sympatric generalist, Neotoma albigula, in response to diets containing different levels of one-seeded juniper (Juniperus monosperma). We found large between species differences in gene expression, as well as large within species differences when specialists fed a low juniper diet (25% juniper) were compared to specialists fed their ecologically relevant level of juniper (70% juniper). We also tested the hypothesis that the specialist relies on less costly phase I detoxification enzymes more than phase II compared to the generalist. Although we found that the specialist had higher cumulative as well as average expression of phase I versus phase II enzymes, the generalist had a similar pattern of expression for phase I versus phase II enzymes.     

Differential expression and activity of catechol-O-methyl transferase (COMT) in a generalist (Neotoma albigula) and juniper specialist (Neotoma stephensi) woodrat

Mammalian herbivores, particularly dietary specialists must have an efficient means to metabolize the high doses of plant secondary compounds they consume. We found previously that Neotoma stephensi, a juniper specialist, upregulated catechol-O-methyl transferase (COMT) mRNA almost seven fold in response to an ecologically relevant diet (70% juniper). To further investigate the relevance of this enzyme with respect to juniper metabolism, we compared the protein expression, activity and kinetics of the two forms of COMT, soluble (S-COMT) and membrane bound (MB-COMT), in the blood, kidneys and liver of N. stephensi on its natural juniper diet to that of N. stephensi fed an experimental diet of 70% juniper as well as a non-toxic control diet under laboratory conditions. In addition, we compared these results to that of Neotoma albigula, a generalist species, which consumes a diet of 25% juniper in the wild. The specialist consuming juniper under both field and laboratory conditions had increased S-COMT expression and activity in their livers and kidneys, and increased S-COMT activity in their blood compared to the specialist and generalist fed the control diet. The specialist showed expression and activity of S-COMT in their kidneys that was as high as or higher than that in their livers. The generalist had an elevated V_max for MB-COMT compared to the specialist that resulted in higher activity for MB-COMT than the specialist despite lower expression of MB-COMT in the generalist's livers and kidneys. This high activity MB-COMT may be in part responsible for differences in the behaviors of the generalist compared to the specialist. We conclude that S-COMT is important in the specialist's ability to consume high levels of juniper.     

Hepatic cytochrome P450 2A6 and 2E1 status in peri-tumor tissues of patients with Opisthorchis viverrini-associated cholangiocarcinoma

Endogenous nitrosation due to chronic inflammation is enhanced in opisthorchiasis and plays a crucial role in the development of cholangiocarcinoma (CCA). Hepatic cytochrome P450 (CYP) family enzymes, especially CYP2A6 and CYP2E1, are involved in the metabolism of procarcinogens; these two enzymes metabolize endogenous nitrosamines to carcinogenic N-dimethylnitrosamine (NDMA). CYP2A6 activity is increased in patients infected with Opisthorchis viverrini. Our aim was to determine whether the expression and function of CYP2A6 and 2E1 in the livers of patients with O. viverrini-associated cholangiocarcinoma (CCA) was altered compared to livers without CCA. Livers of CCA patients (n = 13 cases) showed increased enzyme activities, protein and mRNA levels of CYP2A6 whereas the enzyme activity and protein levels of CYP2E1 were markedly decreased (P < 0.05). CYP2E1 mRNA levels were not altered. Large numbers of inflammatory cells and increased iNOS expression was found in areas adjacent to the tumor. The data provide evidence to support the concept that enhanced CYP2A6 activity and diminished CYP2E1 activity probably involve to the progression of CCA.     

Role of cytochrome P450 2B sequence variation and gene copy number in facilitating dietary specialization in mammalian herbivores

Theory postulates that dietary specialization in mammalian herbivores is enabled by a specialized set of liver enzymes that process the high concentrations of similar plant secondary metabolites (PSMs) in the diets of specialists. To investigate whether qualitative and quantitative differences in detoxification mechanisms distinguish dietary specialists from generalists, we compared the sequence diversity and gene copy number of detoxification enzymes in two woodrat species: a generalist, the white‐throated woodrat (Neotoma albigula) and a juniper specialist, Stephens’ woodrat (N. stephensi). We focused on enzymes in the cytochrome P450 subfamily 2B (CYP2B), because previous research suggests this subfamily plays a key role in the processing of PSMs. For both woodrat species, we obtained and sequenced CYP2B cDNA, generated CYP2B phylogenies, estimated CYP2B gene copy number and created a homology model of the active site. We found that the specialist possessed on average ~5 more CYP2B gene copies than the generalist, but the specialist's CYP2B sequences were less diverse. Phylogenetic analysis of putative CYP2B homologs resolved woodrat species as reciprocally monophyletic and suggested evolutionary convergence of distinct homologs on similar key amino acid residues in both species. Homology modelling of the CYP2B enzyme suggests that interspecific differences in substrate preference and function likely result from amino acid differences in the enzyme active site. The characteristics of CYP2B in the specialist, that is greater gene copy number coupled with less sequence variation, are consistent with specialization to a narrow range of dietary toxins.     

Characterization of Phase I and Phase II hepatic drug metabolism activities in a panel of human liver preparations.

The role of drug metabolism in drug discovery (lead compound selection) and the traditional role of identifying the enzymes involved in biotransformation pathways (reaction phenotyping) have both relied heavily on the availability and use of a human liver bank. The assessment of drug metabolizing enzyme activity and variability in a series of individual human livers is essential when characterizing the enzymes involved in metabolic pathways (i.e. correlation analysis). In this regard, a human liver bank of 21 samples (14 males, six females, and one unknown) was characterized with respect to the activity of several important drug metabolizing enzymes. The total CYP450 content of the livers ranged from 0.06 to 0.46 nmol/mg microsomal protein. The fold variations found in specific enzyme contents were as follows: CYP1A2 (3x), CYP2A6 (21x), CYP2C9 (8x), CYP2C19 (175x), CYP2D6 (18x), CYP2E1 (5x), CYP3A4 (18x), FMO (2.5x), UDPGT (4x), NAT (7x), COMT (5x), ST (5x), TPMT (3x), and GST (2.5x). In general, the fold variation of the Phase II enzymes was lower compared with the Phase I enzymes, with the exceptions of CYP1A2, CYP2E1, and FMO. Similar data were reviewed from other established liver banks and compared with regard to the relative variability observed in drug metabolizing capacities found in this study.     

Ambient temperature influences tolerance to plant secondary compounds in a mammalian herbivore

Growing evidence suggests that plant secondary compounds (PSCs) ingested by mammals become more toxic at elevated ambient temperatures, a phenomenon known as temperature-dependent toxicity. We investigated temperature-dependent toxicity in the desert woodrat (Neotoma lepida), a herbivorous rodent that naturally encounters PSCs in creosote bush (Larrea tridentata), which is a major component of its diet. First, we determined the maximum dose of creosote resin ingested by woodrats at warm (28–29°C) or cool (21–22°C) temperatures. Second, we controlled the daily dose of creosote resin ingested at warm, cool and room (25°C) temperatures, and measured persistence in feeding trials. At the warm temperature, woodrats ingested significantly less creosote resin; their maximum dose was two-thirds that of animals at the cool temperature. Moreover, woodrats at warm and room temperatures could not persist on the same dose of creosote resin as woodrats at the cool temperature. Our findings demonstrate that warmer temperatures reduce PSC intake and tolerance in herbivorous rodents, highlighting the potentially adverse consequences of temperature-dependent toxicity. These results will advance the field of herbivore ecology and may hone predictions of mammalian responses to climate change.     

Regulation of cytochrome P450 gene expression in the olfactory mucosa

The mammalian olfactory mucosa (OM) is unique among extrahepatic tissues in having high levels, and tissue-selective forms, of cytochrome P450 (CYP) enzymes. These enzymes may have important toxicological implications, as well as biological functions, in this chemosensory organ. In addition to a tissue-selective, abundant expression of CYP1A2, CYP2A, and CYP2G1, some of the OM CYPs are also known to have an early developmental expression, a resistance to xenobiotic inducers, and a lack of responsiveness to circadian rhythm. Efforts to fully characterize the regulation of CYP expression in the OM, and to identify the underlying mechanisms, are important for our understanding of the physiological functions and toxicological significance of these biotransformation enzymes, and may also shed unique light on the general mechanisms of CYP regulation. The aim of this mini-review is to provide a summary of current knowledge of the various modes of regulation of CYPs expressed in the OM, an update on our mechanistic studies on tissue-selective CYP expression, and a review of the literature on xenobiotic inducibility of OM CYPs. Our goal is to stimulate further studies in this exciting research area, which is of considerable importance, in view of the constant exposure of the human nasal tissues to inhaled, as well as systemically derived, chemicals, the prevalence of olfactory system damage in individuals with neurodegenerative diseases, and the current uncertainty in risk assessments for potential olfactory toxicants.     

Identification of cytochrome P450 and glutathione-S-transferase genes preferentially expressed in chemosensory organs of the swallowtail butterfly, Papilio xuthus L

The swallowtail butterfly, Papilio xuthus L., feeds exclusively on members of the plant family, Rutaceae. Female butterflies lay eggs in response to specific chemicals contained in their host plants. They perceive a variety of polar compounds as oviposition stimulants through the tarsal chemosensilla of the foreleg by drumming upon the leaf surface. We undertook an expressed sequence tag (EST) analysis to identify the chemosensory-related genes that are expressed in chemosensilla on the tarsus of P. xuthus. Several genes that showed similarity with biotransformation enzymes were identified from the ESTs. Among them, a cytochrome P450 and a glutathione-S-transferase (GST) were preferentially expressed in the chemosensory organs. We have determined the structure of both cDNA and genomic sequences encoding these enzymes and designated the P450 as CYP341A2, a novel member of CYP341A subfamily, and the GST as GST-pxcs1, respectively. We observed a localized expression of CYP341A2 at the base of tarsal chemosensilla by in situ hybridization. These results suggest that these degrading enzymes play a role in the chemosensory reception for host plant recognition.     

Identification and characterization of CYP79D6v4, a cytochrome P450 enzyme producing aldoximes in black poplar (Populus nigra)

After herbivore feeding, poplar trees produce complex volatile blends containing terpenes, green leaf volatiles, aromatics, and nitrogen-containing compounds such as aldoximes and nitriles. It has been shown recently that volatile aldoximes released from gypsy moth (Lymantria dispar) caterpillar-damaged black poplar (Populus nigra) trees attract parasitoids that are caterpillar enemies. In western balsam poplar (P. trichocarpa), volatile aldoximes are produced by 2 P450 monooxygenases, CYP79D6v3 and CYP79D7v2. A gene fragment with high similarity to CYP79D6/7 was recently shown to be upregulated in herbivore-damaged leaves of P. nigra. In the present study we report the cloning and characterization of this gene, designated as CYP79D6v4. Recombinant CYP79D6v4 was able to convert different amino acids into the corresponding aldoximes, which were also found in the volatile blend of P. nigra. Thus, CYP79D6v4 is most likely involved in herbivore-induced aldoxime formation in black poplar.     

Differential expression of Alpha,Mu, and Pi classes of glutathione S-transferases in chemosensory mucosae of rats during development

The expression of three classes of glutathione S-transferases (GSTs), Alpha, Mu, and Pi was investigated in the nasal mucosae of rats during development using immunohistochemical methods. GST Alpha and Mu were first detected in the supranuclear region of sustentacular cells on embryonic days 16. The Bowman's glands expressed differential patterns of immunoreactivity during development, beginning at postnatal day (P) 2 and P6 for Alpha and Mu classes, respectively and being greatest at P11 for both. The acinar cells of vomeronasal glands in the vomeronasal organ expressed Alpha and Mu classes of GSTs from P11 onwards. In the septal organ of Masera, the supranuclear region of sustentacular cells expressed GSTs from P11 with little or no variation during development. In the respiratory mucosa, Alpha and Mu classes of GSTs were detected at the brush borders of ciliated cells and in the acinar cells of posterior septal glands, but not in anterior septal or respiratory glands located on the turbinates. Compared to olfactory mucosa, the changes in immunoreactivity for GSTs were less pronounced in the respiratory mucosa during development. Specific GST Pi immunoreactivity was not detected in the nasal mucosae at any stage of development studied. The occurrence of GSTs in the nasal mucosa, including olfactory, vomeronasal, septal, and respiratory epithelia, suggests that the GSTs are actively involved in the biotransformation of xenobiotics including odorants and pheromones, and may also participate in perireceptor processes such as odorant clearance. In addition, we have developed a working model describing the cellular localization of certain phase I (e.g., cytochrome P-450s) and phase II (e.g., GSTs, -glutamyl transpeptidase) biotransformation enzymes in the olfactory mucosa and their proposed roles in xenobiotic metabolism.