Heterologous biosynthesis of costunolide in Escherichia coli and yield improvement.

Costunolide, the main bioactive compound of the medicinal plant, Radix Aucklandiae, is a sesquiterpene lactone (SL) and has a broad range of biological activities. It is also a precursor of many biologically-active SLs and is a branching point in the biosynthesis of SLs. Here we have reconstituted the costunolide biosynthetic pathway in Escherichia coli by co-expression of three genes (GAS, GAO, LsCOS) involved in costunolide biosynthesis and eight genes involved in converting acetyl-CoA into farnesyl diphosphate from mevalonate pathway. Costunolide production was then detected. By screening and optimization of cultured medium and inducing temperature, costunolide yield was up to 100 mg l(-1) in E. coli.

DNA adduct measurements, cell proliferation and tumor mutation induction in relation to tumor formation in B6C3F1 mice fed coal tar or benzo[a]pyrene.

Coal tar is a complex mixture containing hundreds of compounds, at least 30 of which are polycyclic aromatic hydrocarbons, including the carcinogen benzo[a]pyrene (BaP). Although humans are exposed to complex mixtures on a daily basis, the synergistic or individual effects of components within a mixture on the carcinogenic process remain unclear. We have compared DNA adduct formation and cell proliferation in mice fed coal tar or BaP for 4 weeks with tumor formation in a 2 year chronic feeding study. Additionally, we have analyzed tumor DNA for mutations in the K-ras, H-ras and p53 genes. In the forestomach of mice fed either coal tar or BaP an adduct indicative of BaP was detected, with adduct levels increasing in a dose-responsive manner. K-ras mutations were detected in the forestomach tumors, with the incidence being similar in mice fed coal tar or BaP. These results suggest that the BaP within coal tar is associated with forestomach tumor induction in coal tar-fed mice. DNA adduct levels in the small intestine were not predictive of tumor incidence in this tissue; instead, the tumors appeared to result from compound-induced cell proliferation at high doses of coal tar. K-ras mutations were detected in lung tumors. Since lung tumors were not increased by BaP, coal tar components other than BaP appear to be responsible for the tumors induced in this tissue. H-ras mutations, primarily occurring at codon 61, were the most common mutation observed in liver tumors induced by coal tar. Since this mutation profile is observed in spontaneous hepatic tumors, components in the coal tar may be promoting the expansion of pre-existing lesions.

H- and K-ras mutational profiles in chemically induced liver tumors from B6C3F1 and CD-1 mice.

Liver tumors from mice treated with genotoxic carcinogens often possess mutations in ras protooncogenes, and these sequence alterations in ras frequently reflect the mutational specificity of the carcinogen. Previous studies suggest that the mouse model used for tumor induction may affect ras mutational patterns. In order to explore this possibility, H- and K-ras mutational profiles were established in liver tumors from male B6C3f1 and CD-1 mice administered benzo[a]pyrene (BaP), 6-nitrochrysene (6-NC), and 4-aminobiphenyl (4-ABP). With the exception of 6-NC-induced tumors in B6C3F1 mice, a high proportion of the tumors induced in both types of mice contained ras mutations. In CD-1 mice, 6-NC predominantly induced C-->A mutations in H-ras codon 61 (90% of tumors analyzed), whereas 4-ABP mainly induced A-->T mutations in H-ras codon 61 (50%) and BaP induced both A-->T (27%) and G--> (50%) mutations in H-ras codon 61 and K-ras codon 13, respectively. In B6C3F1 mice, 85% of BaP tumors had G-->C mutations in K-ras codon 13 and 85% of 4-ABP tumors had C-->A mutations in H-ras codon 61, while among 6-NC tumors, only 4% had G-->C mutations in K-ras codon 13 and none had H-ras mutations. Statistical analysis of these results indicates that the patterns of tumor ras mutations induced by BaP in CD-1 and B6C3F1 mice were indistinguishable, while 6-NC and 4-ABP produced different tumor ras profiles in the two mouse models. Published mutational profiles for active metabolites of BaP and 6-NC from in vitro reporter gene systems were inconsistent with both the CD-1 and B6C3F1 tumor ras mutational responses.

Relationships of DNA adduct formation, K-ras activating mutations and tumorigenic activities of 6-nitrochrysene and its metabolites in the lungs of CD-1 mice.

6-Nitrochrysene (6-NC), an environmental pollutant and a potent mouse lung carcinogen, is activated by two major metabolic pathways to yield DNA adducts derived from either trans-1,2-dihydro-1,2-dihydroxy-6-aminochrysene (1,2-DHD-6-AC) or N-hydroxy-6-aminochrysene (N-OH-6-AC). While the former pathway has been shown to be the major activation pathway leading to DNA adducts in mice treated with 6-NC, the potential contribution of the minor nitroreduction pathway to tumorigenicity in this system is not clear. To evaluate the roles of these activation pathways and the resulting DNA adducts in mouse lung tumorigenesis, we studied DNA adduct formation, the induction of tumors and tumor K-ras mutational spectra in the lungs of male CD-1 mice treated with 6-NC and its metabolites. 6-NC, 6-AC and 1,2-DHD-6-AC produced predominantly a single chromatographically identical dG adduct, and 6-nitrosochrysene (6-NOC) gave a single major adduct that was most likely derived from reaction at the C8 position of deoxyadenosine. 6-NC-, 1,2-DHD-6-AC- and 6-NOC-treated mice developed both adenomas and adenocarcinomas in the lung, whereas only lung adenomas were observed in 6-AC-treated animals. K-ras mutations in adenomas resulting from 6-NC and its metabolites were primarily at G:C basepairs in codons 12 and 13, while adenocarcinomas had K-ras mutations distributed between codons 12, 13 and 61, and involved both G:C and A:T basepairs. The K-ras mutational spectra in codons 12 and 13 were similar in both adenomas and adenocarcinomas whereas a higher percentage of mutations at A:T in codon 61 was found in adenocarcinomas. These results support the conclusion that the 1,2-DHD-6-AC-derived adduct is associated with both adenoma and adenocarcinoma formation and is the primary lesion involved in the induction of mouse lung tumors by 6-NC. The major adduct detected after 6-NOC treatment, which is derived from N-OH-6-AC, is apparently less efficient as an inducer of mouse lung tumors and is associated more specifically with adenocarcinoma formation.

Trans-1,2-dihydro-1,2-dihydroxy-6-aminochrysene is metabolized to form a major adduct with deoxyguanosine and produces mutations in the hprt gene of Chinese hamster ovary cells at G:C basepairs.

6-Nitrochrysene can be activated to genotoxic derivatives by two major metabolic pathways: nitroreduction to N-hydroxy-6-aminochrysene, and a combination of ring-oxidation and nitroreduction that involves the intermediate formation of trans-1,2-dihydro-1,2-dihydroxy-6-aminochrysene (6-AC-1,2-dihydrodiol). The DNA adduct formed from this latter pathway was evaluated by reacting individual deoxynucleoside 5'-monophosphates with 6-AC-1,2-dihydrodiol in the presence of liver microsomal enzymes from 3-methylcholanthrene-pretreated rats. Binding was greatest to deoxyguanosine monophosphate and the major deoxyguanosine (dG) adduct co-chromatographed with the single major adduct formed from the microsome-catalyzed reaction of 6-AC-1,2-dihydrodiol with DNA. In order to characterize the mutational changes associated with the 6-AC-1,2-dihydrodiol pathway, we analyzed the mutational spectrum produced by 6-AC-1,2-dihydrodiol in the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene of CHO-K1 cells. cDNA was synthesized from the RNA of 28 6-thioguanine-resistant mutants, the hprt coding region amplified by the polymerase chain reaction, and the DNA products directly sequenced. Twenty independent primary mutations were found: 12 G:C-->T:A transversions, three G:C-->C:G transversions, one G:C-->A:T transition, one A:T-->T:A transversion, two -1 frameshift mutations in sequences containing consecutive guanines, and one 11 bp deletion. All G:C basepair substitutions had the mutated dG on the non-transcribed strand and 86% of the G:C basepair substitutions had one purine 3' to the mutated dG. The pattern of 6-AC-1,2-dihydrodiol-induced basepair substitutions was distinct from the pattern observed in solvent control mutants. These results are consistent with the formation of a promutagenic dG adduct from a metabolite of 6-AC-1,2-dihydrodiol.

32P-postlabelling in studies of arylamine and nitroaromatic hydrocarbon activation and mutagenesis.

Carcinogenic arylamines and nitroaromatic hydrocarbons are chemicals that present occupational health hazards and share pathways of metabolic activation. The 32P-postlabelled DNA adducts formed in Chinese hamster ovary (CHO) cells treated with metabolites from two pathways that are common to the activation of the nitroaromatic hydrocarbon 6-nitrochrysene (6-NC) and the arylamine 6-aminochrysene (6-AC) compared with the spectra of mutations induced at the CHO hprt locus by these were metabolites. 6-Nitrosochrysene (6-NOC), which is reduced by the cells to N-hydroxy-6-AC, formed adducts mainly with deoxyguanosine, but induced mutations primarily through base-pair substitution involving deoxyadenosine. In contrast, 6-AC 1,2-dihydrodiol produced DNA adducts and mutations that mainly involved deoxyguanosine residues. The two major activation pathways for 6-NC and 6-AC thus produce distinct adduct and mutation spectra in CHO cells, and these adduct and mutational spectra are different from those of other arylamines and nitroaromatic hydrocarbons that have been studied.