Coal tar residues produce both DNA adducts and oxidative DNA damage in human mammary epithelial cells.

In the present study we compare the metabolic activation of coal tar, as measured by the production of both DNA adducts and oxidative DNA damage, with that of a single carcinogen that is a constituent of this complex mixture in human mammary epithelial cells (HMEC). We find that a significant level of DNA adducts, detected by 32P-postlabeling, are formed in HMEC following exposure to coal tar residues. This treatment also results in the generation of high levels of oxidative DNA damage, as measured by the production of one type of oxidative base modification, thymine glycols. The amounts of both DNA adducts and thymine varied considerably between the various coal tar residues and did not correlate with either the total amount of polycyclic aromatic hydrocarbons (PAH) or the amount of benzo[a]pyrene (B[a]P) present in the residue. Fractionating the residue from one of the sites by sequential extraction with organic solvents indicated that while the ability to produce both types of DNA damage was contained mostly in a hexane-soluble fraction, a benzene-soluble fraction produced high levels of reactive oxygens relative to the number of total DNA adducts. We find that the total amount of PAH or B[a]P present in the coal tars from the various sites was not a predictor of the level of total DNA damage formed.

Release of markedly increased quantities of prostaglandin D2 from the skin in vivo in humans following the application of sorbic acid.

BACKGROUND AND DESIGN: Sorbic acid is a preservative that can induce cutaneous vasodilation characterized by erythema, urticaria, and stinging when applied topically to humans. Previous studies have suggested that prostaglandins may mediate the vasodilation, but the prostaglandin responsible has not been established. Recently, we have shown that cutaneous erythema similar to that associated with the application of sorbic acid is induced by topical administration of methylnicotinate and is mediated by the release of prostaglandin D2 (PGD2) from the skin. Therefore, we examined whether the cutaneous vasodilation induced by sorbic acid is also mediated by this prostaglandin in humans. RESULTS: Topical application of 1% sorbic acid to the forearms of four human volunteers resulted in 250- to 620-fold increases in levels of PGD2 and 15- to 58-fold increases in levels of the metabolite of PGD2, 9 alpha,11 beta-PGF2, in blood drawn from the antecubital vein draining the treated sites. There were no increases in the release of the other vasodilatory prostaglandins, PGE2 or prostacyclin (PGI2). The release of PGD2 in response to topically applied sorbic acid occurred in a dose-dependent manner and was not accompanied by a release of histamine, suggesting that the release of PGD2 was not from the mast cell. CONCLUSIONS: The cutaneous vasodilation that occurs following the administration of sorbic acid is primarily due to a release of PGD2 from a cellular source in the skin.

Simplified assay for the quantification of 2,3-dinor-6-keto-prostaglandin F1 alpha by gas chromatography-mass spectrometry.

Endogenous prostacyclin production is best assessed by the measurement of its excreted metabolites, of which a major one is 2,3-dinor-6-ketoprostaglandin F1 alpha (2,3-dinor-6-keto-PGF1 alpha). Gas chromatographic-mass spectrometric (GC-MS) assays have been developed for this compound but are cumbersome and time-consuming. We now report a modified assay for the measurement of 2,3-dinor-6-keto-PGF1 alpha employing GC-MS in which sample preparation time is markedly shortened by replacing a number of extraction steps with reversed-phase column extraction and by modifying derivatization procedures. Precision of the assay is +/- 5% and the accuracy is 98%. The lower limit of detection in urine is approximately 15 pg/mg creatinine. Normal urinary levels of this metabolite were found to be 141 +/- 54 pg/mg creatinine (mean +/- S.D.). Urinary excretion of 2,3-dinor-6-keto-PGF1 alpha is markedly altered in situations associated with abnormalities of prostacyclin generation when quantified using this assay. Thus, this assay provides a sensitive and accurate method to assess endogenous prostacyclin production and to further explore the role of this compound in human health and disease.

Free radical-induced generation of isoprostanes in vivo. Evidence for the formation of D-ring and E-ring isoprostanes.

We recently reported the discovery that a series of novel prostaglandin (PG)F2-like compounds (F2-isoprostanes) are produced in vivo independent of the cyclooxygenase as products of free radical-catalyzed lipid peroxidation. F2-isoprostanes are initially formed in situ from arachidonic acid esterified to phospholipids and then released preformed. We have now investigated whether PGD2/E2-like isoprostanes are also produced by rearrangement of the PGG2-like intermediates involved in isoprostane formation. Using a variety of approaches utilizing mass spectrometry, compelling evidence was obtained for the presence of D2/E2-isoprostane-containing phosphospholipids in the liver (85 +/- 33 ng/g of liver) and free D2/E2-isoprostanes in the circulation (215 +/- 90 pg/ml) of rats treated with CCl4 to induce lipid peroxidation. In untreated rats, levels of D2/E2-isoprostanes esterified in liver phospholipids were much lower (0.90 +/- 0.10 ng/g), and free compounds could not be detected in the circulation (< 5 pg/ml). Interestingly, one of the E2-isoprostanes that would be expected to be formed in abundance, 8-epi-PGE2, was found to be a potent renal vasoconstrictor, and these effects could be abrogated by SQ29548, a thromboxane receptor antagonist. Further understanding of the biological consequences of the formation of these novel compounds and factors that influence their formation may provide valuable insights into the pathophysiology of oxidant injury.

Evidence that the F2-isoprostane, 8-epi-prostaglandin F2 alpha, is formed in vivo.

F2-isoprostanes are prostaglandin (PG)F2-like compounds that are produced in vivo as non-enzymatic products of free radical catalyzed peroxidation of arachidonic acid. One F2-isoprostane whose formation should be favored is 8-epi-PGF2 alpha. 8-Epi-PGF2 alpha has been shown to exert potent bioactivity but proof that it is formed in vivo is lacking. Evidence is now presented suggesting that 8-epi-PGF2 alpha is, in fact, formed in vivo by demonstrating that an endogenous F2-isoprostane with a retention time on capillary GC identical with that of 8-epi-PGF2 alpha co-chromatographs through four high resolving HPLC purification procedures with authentic radiolabelled 8-epi-PGF2 alpha.

Improved assay for the quantification of 11-dehydrothromboxane B2 by gas chromatography-mass spectrometry.

Endogenous thromboxane production is best assessed by the measurement of its excreted metabolites, of which 11-dehydrothromboxane B2 (11-dehydro-TxB2) is most abundant. Gas chromatographic-mass spectrometric assays have been developed for this compound but suffer from the presence of co-eluting impurities which make the measurement of 11-dehydro-TxB2 difficult. Furthermore, these assays are often time-consuming. We now report a modified assay for the measurement of this compound employing gas chromatography-mass spectrometry which alleviates the problem of co-eluting impurities primarily through modification of extraction and chromatographic methods. Furthermore, the time to complete the assay is significantly shortened. It is adaptable to both urine and plasma. Precision of the assay is +/- 7% and accuracy is 90%. The lower limit of sensitivity in urine is approximately 20 pg/mg creatinine. Normal levels of urinary excretion of this metabolite were found to be 370 +/- 137 pg/mg creatinine (mean +/- 1 S.D.) and normal plasma levels were found to be 1.5 +/- 0.4 pg/ml (mean +/- 1 S.D.). Urinary excretion of 11-dehydro-TxB2 is markedly altered in situations associated with abnormalities in thromboxane generation when quantified using this assay. Thus, this assay provides a sensitive and accurate method to assess endogenous thromboxane production and to further explore the role of this compound in human disease.

The F2-isoprostane, 8-epi-prostaglandin F2 alpha, a potent agonist of the vascular thromboxane/endoperoxide receptor, is a platelet thromboxane/endoperoxide receptor antagonist.

F2-isoprostanes are a recently discovered series of prostaglandin (PG)F2-like compounds that are produced in vivo in humans by nonenzymatic free radical catalyzed peroxidation of arachidonic acid. One of the compounds that can be produced in abundance by this mechanism is 8-epi-PGF2 alpha. 8-epi-PGF2 alpha is a potent vasoconstrictor in the rat, an effect that has been shown to be mediated via interaction with vascular thromboxane (TxA2)/endoperoxide (PGH2) receptors. In an effort to further understand the biological properties of this prostanoid in relation to its ability to interact with TxA2/PGH2 receptors, we examined its effects on human and rat platelets. At concentrations of 10(-6) M and 10(-5) M, 8-epi-PGF2 alpha induced only a shape change in human platelets and at higher concentrations (10(-4) M) induced reversible but not irreversible aggregation. Both the shape change and reversible aggregation were unaffected by indomethacin but were inhibited by the TxA2/PGH2 receptor antagonist SQ29548. Conversely, 8-epi-PGF2 alpha inhibited platelet aggregation induced by the TxA2/PGH2 receptor agonists U46619 (10(-6) M) and IBOP (3.3 x 10(-7) M) with an IC50 of 1.6 x 10(-6) M and 1.8 x 10(-6) M, respectively. 8-epi-PGF2 alpha also inhibited platelet aggregation induced by arachidonic acid. Similarly, in rat platelets, 8-epi-PGF2 alpha alone induced only modest reversible aggregation but completely inhibited U46619-induced aggregation.