The objectives of this study were to determine whether differences in the size and composition of coarse (2.5-10 micro m), fine (< 2.5 microm), and ultrafine (< 0.1 microm) particulate matter (PM) are related to their uptake in macrophages and epithelial cells and their ability to induce oxidative stress. The premise for this study is the increasing awareness that various PM components induce pulmonary inflammation through the generation of oxidative stress. Coarse, fine, and ultrafine particles (UFPs) were collected by ambient particle concentrators in the Los Angeles basin in California and used to study their chemical composition in parallel with assays for generation of reactive oxygen species (ROS) and ability to induce oxidative stress in macrophages and epithelial cells. UFPs were most potent toward inducing cellular heme oxygenase-1 (HO-1) expression and depleting intracellular glutathione. HO-1 expression, a sensitive marker for oxidative stress, is directly correlated with the high organic carbon and polycyclic aromatic hydrocarbon (PAH) content of UFPs. The dithiothreitol (DTT) assay, a quantitative measure of in vitro ROS formation, was correlated with PAH content and HO-1 expression. UFPs also had the highest ROS activity in the DTT assay. Because the small size of UFPs allows better tissue penetration, we used electron microscopy to study subcellular localization. UFPs and, to a lesser extent, fine particles, localize in mitochondria, where they induce major structural damage. This may contribute to oxidative stress. Our studies demonstrate that the increased biological potency of UFPs is related to the content of redox cycling organic chemicals and their ability to damage mitochondria.

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Ultrafine particulate pollutants induce oxidative stress and mitochondrial damage.

The complexity of tobacco smoke leads to some confusion about the mechanisms by which it causes lung cancer. Among the multiple components of tobacco smoke, 20 carcinogens convincingly cause lung tumors in laboratory animals or humans and are, therefore, likely to be involved in lung cancer induction. Of these, polycyclic aromatic hydrocarbons and the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone are likely to play major roles. This review focuses on carcinogens in tobacco smoke as a means of simplifying and clarifying the relevant information that provides a mechanistic framework linking nicotine addiction with lung cancer through exposure to such compounds. Included is a discussion of the mechanisms by which tobacco smoke carcinogens interact with DNA and cause genetic changes--mechanisms that are reasonably well understood--and the less well defined relationship between exposure to specific tobacco smoke carcinogens and mutations in oncogenes and tumor suppressor genes. Molecular epidemiologic studies of gene-carcinogen interactions and lung cancer--an approach that has not yet reached its full potential--are also discussed, as are inhalation studies of tobacco smoke in laboratory animals and the potential role of free radicals and oxidative damage in tobacco-associated carcinogenesis. By focusing in this review on several important carcinogens in tobacco smoke, the complexities in understanding tobacco-induced cancer can be reduced, and new approaches for lung cancer prevention can be envisioned.

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Tobacco Smoke Carcinogens and Lung Cancer

Steroidogenesis entails processes by which cholesterol is converted to biologically active steroid hormones. Whereas most endocrine texts discuss adrenal, ovarian, testicular, placental, and other steroidogenic processes in a gland-specific fashion, steroidogenesis is better understood as a single process that is repeated in each gland with cell-type-specific variations on a single theme. Thus, understanding steroidogenesis is rooted in an understanding of the biochemistry of the various steroidogenic enzymes and cofactors and the genes that encode them. The first and rate-limiting step in steroidogenesis is the conversion of cholesterol to pregnenolone by a single enzyme, P450scc (CYP11A1), but this enzymatically complex step is subject to multiple regulatory mechanisms, yielding finely tuned quantitative regulation. Qualitative regulation determining the type of steroid to be produced is mediated by many enzymes and cofactors. Steroidogenic enzymes fall into two groups: cytochrome P450 enzymes and hydroxysteroid dehydrogenases. A cytochrome P450 may be either type 1 (in mitochondria) or type 2 (in endoplasmic reticulum), and a hydroxysteroid dehydrogenase may belong to either the aldo-keto reductase or short-chain dehydrogenase/reductase families. The activities of these enzymes are modulated by posttranslational modifications and by cofactors, especially electron-donating redox partners. The elucidation of the precise roles of these various enzymes and cofactors has been greatly facilitated by identifying the genetic bases of rare disorders of steroidogenesis. Some enzymes not principally involved in steroidogenesis may also catalyze extraglandular steroidogenesis, modulating the phenotype expected to result from some mutations. Understanding steroidogenesis is of fundamental importance to understanding disorders of sexual differentiation, reproduction, fertility, hypertension, obesity, and physiological homeostasis.

The molecular biology, biochemistry, and physiology of human steroidogenesis and its disorders.

Quinones represent a class of toxicological intermediates which can create a variety of hazardous effects in vivo, including acute cytotoxicity, immunotoxicity, and carcinogenesis. The mechanisms by which quinones cause these effects can be quite complex. Quinones are Michael acceptors, and cellular damage can occur through alkylation of crucial cellular proteins and/or DNA. Alternatively, quinones are highly redox active molecules which can redox cycle with their semiquinone radicals, leading to formation of reactive oxygen species (ROS), including superoxide, hydrogen peroxide, and ultimately the hydroxyl radical. Production of ROS can cause severe oxidative stress within cells through the formation of oxidized cellular macromolecules, including lipids, proteins, and DNA. Formation of oxidatively damaged bases such as 8-oxodeoxyguanosine has been associated with aging and carcinogenesis. Furthermore, ROS can activate a number of signaling pathways, including protein kinase C and RAS. This review explore...

Role of quinones in toxicology.

Air pollution has been considered a hazard to human health. In the past decades, many studies highlighted the role of ambient airborne particulate matter (PM) as an important environmental pollutant for many different cardiopulmonary diseases and lung cancer. Numerous epidemiological studies in the past 30 years found a strong exposure-response relationship between PM for short-term effects (premature mortality, hospital admissions) and long-term or cumulative health effects (morbidity, lung cancer, cardiovascular and cardiopulmonary diseases, etc). Current research on airborne particle-induced health effects investigates the critical characteristics of particulate matter that determine their biological effects. Several independent groups of investigators have shown that the size of the airborne particles and their surface area determine the potential to elicit inflammatory injury, oxidative damage, and other biological effects. These effects are stronger for fine and ultrafine particles because they can penetrate deeper into the airways of the respiratory tract and can reach the alveoli in which 50% are retained in the lung parenchyma. Composition of the PM varies greatly and depends on many factors. The major components of PM are transition metals, ions (sulfate, nitrate), organic compound, quinoid stable radicals of carbonaceous material, minerals, reactive gases, and materials of biologic origin. Results from toxicological research have shown that PM have several mechanisms of adverse cellular effects, such as cytotoxicity through oxidative stress mechanisms, oxygen-free radical-generating activity, DNA oxidative damage, mutagenicity, and stimulation of proinflammatory factors. In this review, the results of the most recent epidemiological and toxicological studies are summarized. In general, the evaluation of most of these studies shows that the smaller the size of PM the higher the toxicity through mechanisms of oxidative stress and inflammation. Some studies showed that the extractable organic compounds (a variety of chemicals with mutagenic and cytotoxic properties) contribute to various mechanisms of cytotoxicity; in addition, the water-soluble faction (mainly transition metals with redox potential) play an important role in the initiation of oxidative DNA damage and membrane lipid peroxidation. Associations between chemical compositions and particle toxicity tend to be stronger for the fine and ultrafine PM size fractions. Vehicular exhaust particles are found to be most responsible for small-sized airborne PM air pollution in urban areas. With these aspects in mind, future research should aim at establishing a cleared picture of the cytotoxic and carcinogenic mechanisms of PM in the lungs, as well as mechanisms of formation during internal engine combustion processes and other sources of airborne fine particles of air pollution.

Airborne particulate matter and human health: toxicological assessment and importance of size and composition of particles for oxidative damage and carcinogenic mechanisms

The bioactivation of polycyclic aromatic hydrocarbons (PAHs) to their ultimate carcinogenic forms proceeds via the formation of proximate carcinogen trans-dihydrodiols. Previous studies demonstrate...

Expression and characterization of four recombinant human dihydrodiol dehydrogenase isoforms: oxidation of trans-7, 8-dihydroxy-7,8-dihydrobenzo[a]pyrene to the activated o-quinone metabolite benzo[a]pyrene-7,8-dione.

Polycyclic aromatic hydrocarbons (PAHs) are metabolized to trans-dihydrodiol proximate carcinogens by human epoxide hydrolase (EH) and CYP1A1. Human dihydrodiol dehydrogenase isoforms (AKR1C1-AKR1C4), members of the aldo-keto reductase (AKR) superfamily, activate trans-dihydrodiols by converting them to reactive and redox-active o-quinones. We now show that the constitutively and widely expressed human AKR, aldehyde reductase (AKR1A1), will oxidize potent proximate carcinogen trans-dihydrodiols to their corresponding o-quinones. cDNA encoding AKR1A1 was isolated from HepG2 cells, overexpressed in Escherichia coli, purified to homogeneity, and characterized. AKR1A1 oxidized the potent proximate carcinogen (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene with a higher utilization ratio (V(max)/K(m)) than any other human AKR. AKR1A1 also displayed a high V(max)/K(m) for the oxidation of 5-methylchrysene-7,8-diol, benz[a]anthracene-3,4-diol, 7-methylbenz[a]anthracene-3,4-diol, and 7,12-dimethylbenz[a]anthracene-3,4-diol. AKR1A1 displayed rigid regioselectivity by preferentially oxidizing non-K-region trans-dihydrodiols. The enzyme was stereoselective and oxidized 50% of each racemic PAH trans-dihydrodiol tested. The absolute stereochemistries of the reactions were assigned by circular dichroism spectrometry. AKR1A1 preferentially oxidized the metabolically relevant (-)-benzo[a]pyrene-7(R),8(R)-dihydrodiol. AKR1A1 also preferred (-)-benz[a]anthracene-3(R),4(R)-dihydrodiol, (+)-7-methylbenz[a]anthracene-3(S),4(S)-dihydrodiol, and (-)-7,12-dimethylbenz[a]anthracene-3(R),4(R)-dihydrodiol. The product of the AKR1A1-catalyzed oxidation of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene was trapped with 2-mercaptoethanol and characterized as a thioether conjugate of benzo[a]pyrene-7,8-dione by LC/MS. Multiple human tissue expression array analysis showed coexpression of AKR1A1, CYP1A1, and EH, indicating that trans-dihydrodiol substrates are formed in the same tissues in which AKR1A1 is expressed. The ability of this general metabolic enzyme to divert trans-dihydrodiols to o-quinones suggests that this pathway of PAH activation may be widespread in human tissues.

The ubiquitous aldehyde reductase (AKR1A1) oxidizes proximate carcinogen trans-dihydrodiols to o-quinones: potential role in polycyclic aromatic hydrocarbon activation.

Structure-function aspects and inhibitor design of type 5 17β-hydroxysteroid dehydrogenase (AKR1C3)☆

A novel pathway of polycyclic aromatic hydrocarbon metabolism involves the oxidation of non-K-region trans-dihydrodiols to yield o-quinones, a reaction catalyzed by dihydrodiol dehydrogenase (DD). We have recently shown that in isolated rat hepatocytes (±)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-diol) was oxidized by this route to yield benzo[a]pyrene-7,8-dione (BPQ). We now report the disposition of BPQ and its mutagenic and genotoxic properties. Using [3H]BPQ it was found that 30% of the radioactivity was sequestered by rat hepatocytes into the cell pellet. Isolation of hepatocyte DNA provided evidence for a low level of covalent incorporation of BPQ into DNA (30 ± 17 adducts/106 base pairs). Examination of the hepatocellular DNA by agarose gel electrophoresis following treatment with BPQ indicated that extensive fragmentation had occurred. DNA fragmentation was also observed when hepatocytes were treated with BP-diol and this effect was attenuated by indomethacin, a DD inhibitor. Hepatocytes ...

Michael E. Burczynski

Papers

Expression and characterization of four recombinant human dihydrodiol dehydrogenase isoforms: oxidation of trans-7, 8-dihydroxy-7,8-dihydrobenzo[a]pyrene to the activated o-quinone metabolite benzo[a]pyrene-7,8-dione.

The ubiquitous aldehyde reductase (AKR1A1) oxidizes proximate carcinogen trans-dihydrodiols to o-quinones: potential role in polycyclic aromatic hydrocarbon activation.

Structure-function aspects and inhibitor design of type 5 17β-hydroxysteroid dehydrogenase (AKR1C3)☆

Disposition and Biological Activity of Benzo[a]pyrene-7,8-dione. A Genotoxic Metabolite Generated by Dihydrodiol Dehydrogenase†,‡

Metabolic activation of polycyclic aromatic hydrocarbon trans-dihydrodiols by ubiquitously expressed aldehyde reductase (AKR1A1).