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- Yang, Yang
(författare)
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The in vivo metabolism of Benzo(a)pyrene studied by chromatography in combination with mass spectrometry
- 1997
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Polycyclic aromatic hydrocarbons (PAHs) constitute a large class of chemicals with widespread occurrence in the environment. It is essentially impossible to avoid exposure to these substances on a daily basis. Benzo[a]pyrene (BP), a model PAH, is a powerful mutagenic and carcinogenic agent in various experimental systems and is also suspected to be of a significant health risk to humans. To date, the majority of studies on the characterization of BP metabolites has been performed in vitro. The analysis of the integral metabolism of BP occurring in vivo has been hampered due to methodological difficulties in isolating and identifying the myriad of trace BP metabolites formed in vivo owing to their low relative abundance and instability. The thorough characterization of in vivo metabolites of BP in urine from germfree rats, given a single intraperitoneal dose of BP, is described in this thesis. Ion exchange chromatography gives information on the nature of the conjugation. Six fractions were collected: the NEUTRAL FRACTION, containing unconjugated BP, and FRACTIONS I-V containing BP conjugates. The metabolites in NEUTRAL FRACTION were analyzed by HPLC and gas chromatography/mass spectrometry (GC/MS). A trans-11,12-diol was found as a major metabolite of BP in this fraction. Trace levels of metabolites probably related to the well-known activation pathway leading to BP-7,8-dihydrodiol-9,10-epoxide (BPDE) were detected, e.g. the trans-7,8-diol and tetrols. The presence of a high abundance of trihydroxy-BPs may indicate a hitherto unsuspected action of dihydrodiol dehydrogenase (DDH) in the in vivo metabolism of BP. A novel strategy was developed using liquid chromatography/mass spectrometry (LC/MS) to analyze BP conjugates. Bile acid and steroid conjugates in urine, separated by the ion exchange procedure, were used as model compounds. The LC/electrospray MS method was then applied to the analysis of conjugated metabolites of BP (FRACTIONS II-V). The major metabolite in FRACTION II was tentatively characterized as a pentahydroxy-BP or a structural isomer. Conjugates of tetrahydrotrihydroxy-, dihydrodihydroxy- and dihydrotrihydroxy-BPs with N-acetylcysteine and a BP-O-glucuronide were formed in smaller amounts. The predominant compounds were oxygenated at C-7,8,9,10. Metabolites detected in FRACTION m included monohydroxy-BP-O-sulfates, dihydrodihydroxy-BP-O-sulfates and a BP-O,O'-diglucuronide. BP-O,O'-disulfates were found in FRACTION IV. Only a trace level of a tetrahydro-trihydroxy-BP-glutathione conjugate was detected in FRACTION V. The GC/MS and LC/MS strategy was designed to be generally applicable to the analysis of lipophilic endogenous substances, drugs, xenobiotics and their metabolites in biological matrices.
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- Abdel-Halim, SM, et al.
(författare)
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Mutations in the promoter of adenylyl cyclase (AC)-III gene, overexpression of AC-III mRNA, and enhanced cAMP generation in islets from the spontaneously diabetic GK rat model of type 2 diabetes
- 1998
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Ingår i: Diabetes. - : American Diabetes Association. - 0012-1797 .- 1939-327X. ; 47:3, s. 498-504
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Tidskriftsartikel (refereegranskat)abstract
- Glucose-induced insulin release is decreased in the spontaneously diabetic GK rat, a nonobese rodent model of type 2 diabetes. Forskolin restores the impaired insulin release in both the isolated perfused pancreas and isolated islets from these rats (Abdel-Halim et al., Diabetes 45:934-940, 1996). We demonstrate here that the insulinotropic effect of forskolin in the GK rat is due to increased generation of cAMP and that it is associated with overexpression of adenylyl cyclase (AC)-III mRNA and gene mutations. The AC-III mRNA overexpression was demonstrated by in situ hybridization using oligonucleotide probes binding to different regions of the rat AC-III mRNA. It was associated with the presence of two point mutations identified at positions -28 bp (A --> G) and -358 bp (A --> C) of the promoter region of the AC-III gene and was demonstrable in both GK rat islets and peripheral blood cells. Transfection of COS cells with a luciferase reporter gene system revealed up to 25-fold increased promoter activity of GK AC-III promoter when compared with normal rat promoter (P < 0.0001). In conclusion, forskolin restores the impaired insulin release in islets of the GK rat through enhanced cAMP generation. This is linked to overexpression of AC-III mRNA in GK islets due to two functional point mutations in the promoter region of the AC-III gene.
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