The mobile phase consisted of ACN and water containing 0.1% trifluoroacetic acid beginning with 65% and increasing to 100% over 11 min at 1 mL/min. A final wash at 65% ACN for 6 min eluted interfering materials. The tR values for HCHO-DNPH and benzaldehyde-DNPH were verified with analytical standards. Formaldehyde dehydrogenase was used to determine if the HCHO produced was bound or free. Microsomal incubations after 1 h were treated with 0.025 units FDH , 40 mM NAD+ and 80 mM reduced glutathione followed by a 20-min incubation at 37°C then analysis for HCHO as above. Some N-methylol metabolites such as diuron or monuron N-methylol can be detected after methylation to form a N-methoxymethyl derivative. Following the Suzuki procedure, TMX, dm-TMX or diuron-microsomal-NADPH incubations were extracted four times with ethyl ether which was then evaporated under N2 at 25°C to 1 mL and 100 µL of methanol and 20 µL of concentrated sulfuric acid were added. After shaking for 1 min at room temperature, reactions were extracted with ice-cold water and the ether layer recovered. The aqueous fraction was further extracted with ethyl ether. The ether fractions were combined, evaporated to dryness and analyzed as in section 2.2.5 for methylated N-methylol intermediates, but none were identified.TMX is a hepatotoxicant and hepatocarcinogen in mice and its metabolite, dm TMX, is also hepatotoxic in mice with its toxicity exacerbated by dm-CLO as an iNOS inhibitor. Importantly, these unfavorable toxicological features are not evident in rats,macetas 20 litros raising the question of whether mice or rats are the better model for humans.
Comparative metabolism may be a factor since mouse liver microsomes form dm-TMX and dm-CLO much more efficiently than rat or human liver microsomes. TMX was initially categorized as a “likely human carcinogen” based on the mouse model but this was modified to “not likely to be carcinogenic to humans” based on species differences in metabolism. Initial preliminary studies focused on the in vitro inhibition of two isoforms of NOS by dm-CLO. Reducing the formation of nitric oxide via NOS inhibition is known to enhance hepatotoxicity of other toxicants and may explain the hepatotoxicity observed in TMX-treated mice. However, the tentative conclusion is that dm-CLO does not potently inhibit either iNOS or nNOS in vitro. Therefore the focus of further studies turned to analyzing the formation of other reactive metabolites from TMX and dm TMX. TMX is the only one of the seven commercial neonicotinoids to induce hepatotoxicity or hepatocarcinogenicity in mice or rats. The unique structural feature of TMX and its hepatotoxic metabolite dm-TMX is the oxadiazinane moiety, which is a potential source of HCHO and N-methylol metabolites. In this first study of HCHO as a neonicotinoid CYP metabolite we find that of all commercial neonicotinoids TMX and dm-TMX are the most efficient HCHO generators and much more so with mouse than rat or human CYPs. Our results on species differences in HCHO liberation fully agree with the findings of Green et al.on dm-TMX, CLO and dm-CLO formation from the rest of the molecule. The observed species differences in metabolism of TMX or dm-TMX are likely due to substrate specificity and expression differences of various CYP enzymes, with mice having the highest number of CYP genes compared to rats and humans.
The present study therefore confirms the preference for the rat over the mouse model in TMX human risk assessment. The hepatotoxicant/ hepatocarcinogen candidates from TMX and dm-TMX metabolism are HCHO and N-methylol intermediates. HCHO is a known human carcinogen. To test if free HCHO was formed, FDH was added to TMX- mouse microsomal incubation reactions. Based on FDH-induced HCHO loss, most of the HCHO formed by CYPs from TMX was free, but the remaining HCHO could be protein bound or released from N-methylol intermediates during analysis. A similar result was obtained for HCHO liberated from the NCH2OCH3 moiety of alachlor under comparable conditions. Attempts to detect N-methylol metabolites in the liver of TMX-treated mice were unsuccessful in vivoas well as in vitropossibly due to instability on formation and analysis. The same applies to in vivo detection of HCHO which is very bioreactive. Although not detailed here, white blood cells from the same groups of TMX-treated and control mice were analyzed for DNA-protein crosslinks induced by HCHO production from TMX using the method of Zhitkovich and Costa. These attempts were unsuccessful with the assay method having low sensitivity. Many compounds including pesticides proposed or established to be carcinogens yield N-methylol metabolites some of which may contribute to their toxicity. The inability to detect N-methylol metabolites from alachlor or HMPA in our studies agree with previous literature indicating the difficulty of their analysis due to high reactivity and short half-lives. The candidate N-methylol intermediates from TMX and dm-TMX were therefore synthesized by reaction of CLO and dm-CLO with HCHO and HCO2H. Although individual intermediates were not isolated and characterized, the CLO and dm-CLO reactions with HCHO in HCO2H appear to give two mono-N-methylols and three further addition compounds with m/z equivalent to the addition of two methylols per molecule as the reaction proceeds ultimately ending up as TMX and dm-TMX.
The proposed mono-N methylols from synthesis were found to be stable not only to extraction and LC/MS but also to an additional incubation with mouse liver microsomes. This creates an apparent contradiction wherein the synthetic N-methylols are adequately stable for study but the proposed CYP-formed N-methylols are not observed. This anomaly is rationalized by the different mechanisms and environment of N-methylol formation in the chemical synthesis and enzymatic CYP systems. Perhaps the N-methylols as they are enzymatically formed further react in the CYP active site containing ferrous iron,maceta 40 litros cysteine thiol and imidazole N-H. Cannabis sativa has a long history of cultivation for a variety of uses including food, fibre, medicine, and recreational drugs. Cannabis produces many different secondary compounds such as cannabinoids, flavonoids, stilbenoids, alkaloids, lignanamides, and phenolic amides. D9 -Tetrahydrocan nabinolic acid , a product of the cannabinoid class, is the primary psychoactive agent. This compound is produced as an acid in the glandular trichomes of in- flflorescence bracts and undergoes decarboxylation with age or heating to D9 -tetrahydrocannabinol. Cannabis cultivars differ substantially in economic traits that range from marijuana, arguably the most widespread illicit drug, to hemp fibre derived from the stems of the plant. Marijuana consists of the dried female inflorescences in which the quantity of THC exceeds that of cannabidiol, and potency varies among cultivars by several orders of magnitude. Marijuana cultivars are known to have THC levels exceeding 2–24% of inflorescence dry weight whereas hemp cultivars produce substantially less THC but rather high levels of CBD. THCA and CBDA share the same bio-synthetic pathway except for the last step in which THCA synthase and CBDA synthase produce THCA or CBDA, respectively.
Recent evidence suggests that the genes encoding the two synthases are allelic. CBD and THC are enatiomers, but only THC elicits psychotropic effects, whereas CBD may mediate anti-psychotropic effects , a difference highlighting the stereo-selectivity of receptors in the human body that bind these compounds. Although classified as a drug without therapeutic value in the United States, ingestion of THC is widely regarded as having effects including pain relief and appetite stimulation, that may, among other things, increase the tolerance of cancer patients to chemotherapy. Dronabinol, a synthetic analogue of THC, is approved for use as an appetite stimulant in the United States as a Schedule III drug. Cesamet , another synthetic analogue, is used as an anti-emetic for patients undergoing cancer therapy. The natural product Sativex is approved for use in the UK and is derived from Cannabis cultivars containing both THC and CBD, and is used to treat pain symptoms associated with multiple sclerosis. Compounds from Cannabis sativa are of undeniable medical interest, and subtle differences in the chemical nature of these compounds can greatly influence their pharmacolog ical properties. For these reasons, a better understanding of the secondary metabolic pathways that lead to the synthesis of bio-active natural products in Cannabis is needed. Knowledge of genetics underlying cannabinoid bio synthesis is also needed to engineer drug-free and distinctive Cannabis varieties capable of supplying hemp fibre and oil seed. In this report, RNA from mature glands isolated from the bracts of female inflorescences was converted into cDNA and cloned to produce a cDNA library. DNA from over 2000 clones has been sequenced and characterized. Candidate genes for almost all of the enzymes required to convert primary metabolites into THCA have been identified. Expression levels of many of the candidate genes for the pathways were compared between isolated glands and intact inflorescence leaves.Seeds from the marijuana cultivar Skunk no. 1 were pro vided by HortaPharm BV and imported under a US Drug Enforcement Administration permit to a registered controlled substance research facility. Plants were grown under hydroponic conditions in a secure growth chamber yielding cannabinoid levels in mature plants as reported in Datwyler and Weiblen. Approximately 5 g of tissue was harvested from mature female inflorescences 8 weeks after the onset of flowering. Tissue was equally distributed into four 50 ml tubes containing 20 ml phosphate buffered saline as de scribed by Sambrook et al. , but made with all potassium salts and mixed at maximum speed with a Vortex 2 Genie for four repetitions of 30 s mixing followed by 30 s rest on ice, for a total of 2 min of mixing. Material was sieved through four layers of 131 mm plastic mesh and the flow-through was split into two 50 ml tubes and spun in a centrifuge for 30 s at 500 rpm. Supernatants were decanted and pellets were resuspended in PBS. The suspensions were combined into one tube and pelleted as before. The resulting pellet was diluted into 100 ll of PBS. Five ll were used for cell counting with a haemocytometer, and the total suspension was estimated to contain 70 000 intact glands. Plant residue was incinerated by a DEA-registered reverse distributor. Quantitative reactions were performed as described previously using primers listed in Supplementary Table 4B at JXB online. Equivalent quantities of RNA isolated from glands and inflorescence associated leaves were used to generate the respective single stranded cDNAs. qPCR reactions containing equal quantities of gland or leaf cDNA were run in duplicate along with reactions containing standards consisting of 100-fold se quential dilutions of isolated target fragments, on a Light cyler qPCR machine. Lightcycler software was used to generate standard curves covering a range of 106 to which gland and leaf data were compared. Two biological replicates were used to generate the means and standard deviations shown in Supplementary Table 4A at JXB online. These values were used to compute the gland over leaf ratios and P-values shown in Supplementary Table 4A at JXB online. Raw relative expression data, means, standard deviations, P-values from gland versus leaf t tests, qPCR primer sequences, and representative real-time qPCR tracings are shown in Supplementary Table 4A at JXB online.Anatomical study revealed that glands located on mature floral bracts of female plants are the site of enhanced secondary metabolism leading to the production of THCA and other compounds in Cannabis sativa. These glands are located on multicellular stalks and typically are composed of eight cells.The capsule contains exudates derived from the gland cells. The weakly attached glands can easily be separated from the bracts and purified as shown in Fig. 1E and F. An EST library was constructed using RNA isolated from purified glands. Over 100 000 ESTs were cloned. Plasmid DNA was isolated and sequenced from over 2000 clones. Because of the directed orientation of cDNA insertion, sequences are expected to represent the coding strand. After the removal of vector only, poor quality sequences, and sequences obviously originating from organelles or ribosomal RNA, the remain ing sequences were clustered into 1075 unigenes. Overall, 111 of the unigenes were contigs containing two or more closely related ESTs. Only 14 contigs lacked a similar sequence in the NCBI database. Nine hundred and sixty four of the ESTs were only found once and of these 710 were similar to sequences in the NCBI database.