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The major polycyclic aromatic hydrocarbon inducible-cytochrome P4501A1 gene (CYP1A1) is presumed to be important in pulmonary carcinogenesis and toxicology because its product, the cytochrome P4501A1-dependent (CYP1A1-dependent) monooxygenase, transforms selected xenobiotics (including polycyclic aromatic hydrocarbon procarcinogens in cigarette smoke) to potent carcinogenic metabolites. CYP1A1 messenger RNA (mRNA) expression has not, however, been previously demonstrated in human pulmonary tissue. This report defines CYP1A1 gene expression in normal lung tissue and primary pulmonary carcinoma tissue obtained at thoracotomy from 56 patients with lung cancer. When Northern blot hybridization analyses were performed, 17 of 19 (89%) and zero of five (0%) samples of normal lung tissue from active cigarette smokers and nonsmokers, respectively, expressed the normal 2.8-kilobase CYP1A1 mRNA. In addition, a time-dependent decrease in expression of the CYP1A1 gene was noted in normal lung tissue from individuals who were former smokers, with a decrease in expression occurring as early as 2 weeks following cessation of cigarette smoking. Expression became undetectable in all patients who had stopped smoking more than 6 weeks prior to study. When CYP1A1 gene expression was evaluated in lung cancers, mRNA levels were detectable in one of four (25%) tumors from nonsmokers; two of 24 (8%) tumors from former smokers; and seven of 15 (47%) tumors from cigarette smokers. In addition, an approximately 10-kilobase CYP1A1 RNA species, which was not detectable in normal lung tissue, was observed in five of ten (50%) of the lung cancers that expressed the CYP1A1 gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Histamine and certain cyclooxygenase products of arachidonic acid have been implicated as mediators of inflammation and are potent constrictors of human airways. Because asthma may represent manifestations of chronic inflammation of the airways, the levels of histamine and six prostanoid mediators were measured in airway fluids obtained by bronchoalveolar lavage (BAL) of 12 normal, 11 allergic rhinitic, and 15 asymptomatic, allergic asthmatic subjects. Simultaneous profiling of prostanoid mediators in individual samples was performed using gas chromatography-mass spectrometry. Levels of PGD2, 9 alpha,11 beta-PGF2 and PGF2 alpha were 12 to 22 times higher in asthmatic than in normal subjects (p less than 0.01), with concentrations in airway fluids of asthmatic subjects after correction for dilution of 3.8, 0.5, and 1.4 nanomolar, respectively. Levels of PGD2 and 9 alpha,11 beta-PGF2 were increased nearly tenfold in asthmatic subjects compared with those in rhinitic subjects (p less than 0.01), distinguishing the subjects with lower airway disease from those with another atopic condition. Histamine levels were increased fourfold in asthmatic subjects compared with those in normal subjects (p less than 0.001); however, similar increases were found in rhinitic subjects. We conclude that elevated levels of multiple mediators with potent bronchoconstricting activity are present in the airways of subjects with mild asthma, indicating that even mild disease is associated with evidence of airway inflammation. The interactions of bronchoconstricting mediators and airway inflammation may play important roles in the pathogenesis of asthma.

The prostaglandins, leukotrienes and related eicosanoids have been implicated as mediators in human malignant disease, particularly in cellular events related to tumor metastasis, cell proliferation, tumor promotion and host immunoregulation (1–23). There is substantial evidence that human tumor cells may synthesize significant quantities of prostaglandins. Elevated production of prostaglandin E2 (PGE2) has been demonstrated in lung cancer patients in vivo (24,25). Other studies have shown that prostanoid biosynthesis is elevated in human tumor tissues in comparison with production in normal human tissues (26–28) and that cultured human tumor cells synthesize significant quantities of prostanoids (29–32). In the present studies, the profiles of prostanoid biosynthesis from endogenous arachidonic acid in 55 established cell lines derived from human tumors of the colon, lung, prostate, ovary, kidney, and the central nervous system were determined. The objective of these studies was the determination of PGH synthase activity in diverse histological classes of human tumor cells in order to discern whether fatty acid cyclooxygenase metabolism of arachidonic acid may be uniquely characteristic of certain histological classes of human tumors.

4-Ipomeanol (IPO) is a pulmonary-specific toxin that is metabolically activated by a cytochrome P450 pathway in lung tissue. In this study, IPO metabolism, as determined by measurement of [14C]IPO covalent binding, was evaluated in a diverse sampling of 18 established, human lung cancer cell lines as well as in normal lung tissue and primary lung carcinoma tissue obtained at the time of thoracotomy from 56 patients with lung cancer. [14C]IPO covalent binding in lung cancer cell lines ranged from 248 to 1,047 pmol of bound [14C]IPO per milligram of protein per 30 minutes (mean +/- SE = 547 +/- 62.2). IPO metabolism in normal lung tissue ranged from 12 to 2,007 pmol of covalently bound [14C]IPO per milligram of protein per 30 minutes (mean +/- SE = 549 +/- 60). In lung cancer tissue, values ranged from 0 to 2.566 pmol of covalently bound [14C]IPO per milligram of protein per 30 minutes (mean +/- SE = 547 +/- 60, P greater than .3). When patients were divided into smokers and current non-smokers (no tobacco products smoked for greater than 6 mo), no effects of cigarette smoking were observed for either normal lung tissue or lung tumor tissue (P greater than .1 in all instances). A wide range of IPO metabolic activity was observed among different histological classifications of lung cancer cell lines and of fresh lung cancer tissues. IPO metabolism was simultaneously compared in normal lung tissue and lung cancer tissue from individual patients, but no positive correlation was observed (r = .10; P greater than .30). The results clearly demonstrate a wide range of IPO metabolism in both normal and lung cancer cells and indicate that a wide diversity of human lung cancers possess the metabolic enzyme system(s) necessary for the bioactivation of IPO to a potentially cytotoxic intermediate. Therefore, the continued exploration for any possible therapeutic potential of IPO in patients with lung cancer appears warranted.

Aryl hydrocarbon hydroxylase was measured in cultured human lymphocytes induced with benzathracene and in pulmonary alveolar macrophages induced in situ in cigarette smokers. Considered separately, neither lymphocyte AHH nor macrophage AHH levels were distinctly different in either noncancer or lung cancer patients. Considered simultaneously, lymphocyte and macrophage AHH levels are quite different in noncancer and lung cancer patients. The lung cancer patient group was seen to contain a significantly higher percentage of persons with high levels of AHH than did an age-matched group of noncancer patients, (P less than 0.001), when more than one tissue was assayed to determine the individual's enzyme levels.

A component capable of binding benzo(a)pyrene was measured in plasma from cigarette smokers and nonsmokers. This plasma fraction was found to have a high specificity of binding to benzo(a)pyrene, bound benzanthracene competitively with benzo(a)pyrene, and was positively correlated (r = 0.861, p less than 0.001) with the capacity of the individual subject's lymphocytes to be induced for AHH activity in culture. An inverse correlation (r = -0.957, p less than 0.001) between the presence of the plasma component in lung cancer patients and the capacity of lung cancer patients' lymphocytes to be induced in culture is unexplained at this time. A benzo(a)pyrene-binding fraction was not found in induced or uninduced cultured lymphocytes from smokers or nonsmokers, or in homogenates of lung excisional tissue from smokers with or without primary lung cancer.

Free alveolar macrophages (FAMs) obtained by bronchoalveolar lavage from healthy nonsmoking volunteers were incubated with varying concentrations (0–300 μg/ml) of Mt. Saint Helens volcanic ash obtained from either Portland, Oregon, or Pullman, Washington, to assess the cytotoxic effects of the ash on human lung cells. Trypan dye exclusion techniques were employed for assessment of cell viability. Following the initial 24 hour culture with the Portland ash samples, decreased viability was observed at all ash concentrations (P < 0.001 in all instances), and further decreases in viability were noted at 48 and 72 hours for all concentrations of ash tested (P < 0.001 in all instances). When the Pullman, Washington, ash sample was evaluated, a decrease in cell viability was noted for the 300 μg/ml concentration (P < 0.017) after the initial 24 hours in culture. Further decreases in cell viability were noted only when cells were cultured for longer time intervals (48 and 72 hours) (P < 0.05 in all instances). Differences in cellular response to the 2 ash samples were further investigated by exposing FAMs from a single individual to the 2 different types of ash. These studies demonstrated similar cytotoxic effects of the 2 ash samples at all concentrations and times tested (P < 0.30 in all instances) with the exception of the 100 μg/ml concentrations at 72 hours (P < 0.020). These data suggest that the differences observed between the 2 types of ash in the independent studies are probably related to interindividual variation in FAM response to the ash rather than to differences in the cytotoxicities of the 2 ash samples. Cytotoxicity of the volcanic ash was also compared with other environmentally relevant airborne particulates, such as amosite and chrysotile asbestos, as well as amorphous and crystalline silica. These results demonstrated an intermediate cytotoxic effect of the ash between innocuous amorphous silica and the very cytotoxic chrysotile asbestos. The affinity for volcanic ash to adsorb tritiated benzo(a)pyrene (3H-BaP) was also compared with that of amorphous silica and amosite asbestos. These studies demonstrate that volcanic ash has intermediate adsorption qualities (4.3 ± 0.1; pmoles 3H-BaP adsorbed/μg particulate ± SD) between those of amorphous silica (1.9 ± 1.0) and amosite asbestos (7.8 ± 1.2) (P < 0.05 in all instances). These data suggest volcanic ash exhibits moderate biological properties compared with those of other environmentally important airborne particulates. Whether in vitro studies reflect in vivo response of human lung cells to the ash cannot be determined at this time, and follow-up of assessment of individuals exposed to the ash will be required to assess its long-term effects on pulmonary tissue.

In this paper we will attempt to provide some explanations for the organ selectivity of four different pneumotoxicants (monocrotaline, naphtalene, 3-methylindole, and 4-ipomeanol)

Human mitogen-stimulated lymphocytes, cultured in the presence of amosite asbestos (AS), demonstrated a slight increase in aryl hydrocarbon hydroxylase (AHH) activity compared with non-induced (control) cultures (P = 0.005). A much greater increase in enzyme activity occurred following addition of the inducers benzanthracene (BA) or cigarette tars (CT) to cell cultures (P less than 0.001 in both instances). Significant enzyme induction also occurred when AS fibers were first preincubated with CT or BA, washed with acetone, then added to lymphocyte cultures (P less than 0.003 in all instances). This increase in AHH activity was not as great, however, as the induction observed when BA or CT was added to cell cultures. No further increase in enzyme activity was noted when AS and CT or AS and BA were simultaneously added to culture lymphocytes (P greater than 0.070 in all instances). The results demonstrate that polycyclic aromatic hydrocarbons (PAH), such as BA and other components of CT, are adsorbed and transported by amosite AS particles. These AS-PAH complexes are capable of inducing AHH in cultured human lymphocytes.

Severe acidosis associated with acetazolamide therapy is rare. We report the first case in which plasma and whole blood acetazolamide concentrations were measured. A 61 year-old patient receiving oral acetazolamide for treatment of glaucoma presented with a 7 day history of declining mental status. The patient was lethargic and oriented only to name. The respiratory rate was 36 per minute in a Kussmaul pattern with arterial blood gases revealing a pH of 7.23, pO2 68 mmHg, paCO2 14 mmHg and bicarbonate 6 mEq/L. Serum creatinine was 3.1 mg%, Cl 126 mEq/L, and anion gap 15. Urine pH was 6.0. Infection and other causes of acidosis and bicarbonate loss were excluded, and he was discharged with normal mental status and improving acid-base balance 18 days after admission. Acetazolamide concentrations four days after the last dose were 26.38 mcg/ml and 38.84 mcg/ml in serum and whole blood, respectively. The serum half-life was 34 hours, compared to a range of 1.5 to 6 hours in subjects with normal renal function. Monitoring acetazolamide concentrations may be useful in adjusting dosage and preventing toxicity in patients with decreased renal function.

The cytochrome P450 (CYP) systems catalyze the metabolic transformation of a wide variety of xenobiotics including procarcinogens present in cigarette smoke condensate as well as atmospheric pollutants. The CYP1A1 isoenzyme is of particular interest because it has been implicated as a risk factor in the etiology of lung cancer in heavy cigarette smokers. The identification and expression of the structural CYP1A1 gene in either normal human lung or lung cancer cells has not been reported. Because of its potential significance in human lung cancer, we investigated the expression of the CYP1A1 structural gene in 24 established human lung cancer cell lines including 15 non-small cell (eight adenocarcinomas, three large cell undifferentiated carcinomas, two bronchioloalveolar cell carcinomas, and two squamous cell carcinomas) and nine small cell lung carcinomas. CYP1A1 mRNA was detected in 14 of 15 (93%) of the non-small cell lung carcinoma cell lines examined following 24-hour treatment with benz[a]anthracene (BA) and in nine of 15 (60%) of the non-small cell lines cultured without an inducer in the medium. When the small cell lung cancer lines were evaluated for CYP1A1 gene expression, two of nine (22%) expressed detectable CYP1A1 mRNA in both BA-induced cell cultures and constitutive (control) cultures. A positive correlation was noted between BA-induced CYP1A1 mRNA levels and the corresponding aryl hydrocarbon hydroxylase activity expressed as absolute BA-induced enzyme activity (r = 0.74; P less than .01; n = 24), which further demonstrated that CYP1A1 mRNA expression reflects CYP1A1 enzyme activity in the individual cell lines. These observations represent the first known demonstration of constitutive (non-induced) CYP1A1 gene expression in human cells and suggest altered regulation of the CYP1A1 gene in selected lung cancer cell lines. These human pulmonary carcinoma cell lines, which have documented regulatory defects, could be useful for further identification of the mechanisms associated with CYP1A1 gene regulation.

Aryl hydrocarbon hydroxylase induction was studied in cultured peripheral blood lymphocytes and pulmonary alveolar macrophages from 15 smokers and 8 nonsmokers with a variety of pulmonary diseases. Enzyme levels in lymphocytes from cigarette smokers cultured in medium without an inducing agent were 57±6 mU/106 cells (mean±SEM), while enzyme levels in lymphocytes from nonsmokers were 20±2 mU/106 cells (P < 0.001). When lymphocytes were cultured in the presence of the inducing agent, benzo-(a)anthracene, enzyme activity was increased to 168±23 mU/106 cells in smokers' cells and 99±22 mU/106 cells in lymphocytes from nonsmokers (P < 0.04). When noninduced enzyme values in cultured macrophages were compared, smokers' cells had enzyme levels of 45±5 mU/106 cells, whereas nonsmokers had enzyme activity of 24±2 mU/106 cells (P < 0.002). However, pulmonary macrophages from smokers or nonsmokers, cultured in the presence of benzo(a)-anthracene, had similar levels of induced enzyme activity (P > 0.1). A positive correlation was observed for nonsmokers (r = 0.596, P > 0.1

5S ond study consisted of 70 golden syrian hamsters: 54 experimental and 16 control animals. The implantation of the thread in the trachea induced a regenerative basal cell hyperplasia of the epithelium. We Ibund squamous metaplasia and progressive intraepithelial neoplasia (lEN) prior to the development of squamous cell carcinoma (CA). In the experimental guinea pigs, only one invasive CA was found at 265 days postoperation. In the experimental hamsters, the first CA was seen at 55 days postoperation, and 65% of the animals sacrificed between 120 and 250 days developed CA. Most of the hamsters with CA also developed spindle cell tumors in the tracheal stroma. The control hamsters and guinea pigs did not develop lEN, and the mature respiratory epithelium was reconstituted. We conclude that this method reliably produces localized, readily accessible preneoplastic and neoplastic lesions in the trachea of hamsters and, to a lesser extent, in that of guinea pigs. This model of squamous cell bronchogenic carcinoma should prove useful in the study of tumor ultrastructure, the immunologic response to cancer, and the relationship of diet to cancer.

Asbestos bodies represent the product of the macrophage's attempt to detoxify inhaled asbestos fibers. The process of asbestos body maturation was examined by scanning electron microscopy of material isolated from lungs of former asbestos workers. The results suggest that the progression from a membrane-bound, smooth coating of the fiber, to the typically beaded form may be caused by cracking and subsequent erosion due to the inspiratory and expiratory forces of the lung on the asbestos body.

Human pulmonary alveolar macrophages were used to quantitate the cytotoxic effect of surface-altered chrysotile asbestos. Little difference was observed in mortality between chrysotile asbestos that was surface-treated to a 42% extent by a hydrophobic organosilane or untreated chrysotile. Little or no effect on mortality was observed when human pulmonary alveolar macrophages were cultured with untreated chrysotile or acid-leached asbestos in the presence of 10 mM dipalmitoyl lecithin. However, when human pulmonary alveolar macrophages were cultured with a hydrophobically-treated (to a 42% or 95% extent) chrysotile asbestos in the presence of 10 mM dipalmitoyl lecithin, a statistically significant decrease in mortality was observed compared to untreated chrysotile. No mutagenic activity was observed when V79 cells were cultured with acid-leached, or 42% hydrophobically-treated chrysotile asbestos, even when human pulmonary alveolar macrophages were included as an activation source. The 95% hydrophobically-treated and acid-leached chrysotile also exhibited decreased binding of benzo[a]pyrene compared to untreated chrysotile asbestos.

Summary Cigarette smoking is associated with alterations in pulmonary alveolar macrophages (PAMs), including increased cytoplasmic inclusions and induction of the aryl hydrocarbon hydroxylase (AHH) system. Nonpigmented PAMs from nonsmokers were able to ingest and accumulate pigment from lysed PAMs of smokers, however, this pigment did not induce AHH activity in either PAMs or peripheral lymphocytes. In contrast, the cigarette tars significantly induced AHH levels in PAMs and in peripheral lymphocytes from either nonsmokers or smokers. This provides further evidence that components in cigarette smoke can explain the in vivo induction of AHH documented in cells from smokers.

The use of reversed-phase thin-layer chromatography for the separation of benzo[a]pyrene metabolites has been investigated. Two systems are described for the separation of the major metabolites of benzo[a]pyrene, including sulfate and glucuronide conjugates.

Methods for the profiling of prostaglandin F2 alpha (PGF2 alpha), prostaglandin D2 (PGD2), prostaglandin E2 (PGE2), thromboxane B2 (TXB2) and 6-keto-prostaglandin F1 alpha (6KPGF1 alpha) biosynthesis in tissue samples of clinical origin by capillary gas chromatography-negative ion chemical ionization mass spectrometry (CGC-NICIMS) are detailed. Aliquots (25 microliter 1) of incubates (1 ml volume) of human lung carcinoma and normal human lung tissue fragments (total protein content = 0.2 to 2.0 mg) were derivatized for vapor phase analysis in the presence of 0.75 to 1.60 ng of tetradeuterated analogs of PGE2, PGF2 alpha and 6KPGF1 alpha without prior extraction and/or chromatography. The derivatized analytes and internal standards were detected by simultaneous monitoring of ions at six different masses characteristic for each of the derivatized prostanoids. The inter-sample and intra-sample coefficients of variation for the assay method were typically less than 12%. The analysis of biological samples was completed with less than 2.5% of each derivatized sample per injection. The samples were of adequate purity for the identification and quantitation of each of the eicosanoids. The methods described in this report are highly selective and highly sensitive with detection limits of 0.1 to 0.2 picograms per injection. The analytical procedures provide the basis for comparisons of the qualitative and quantitative profiles of prostaglandin biosynthesis and should be adaptable for use in a variety of biological and clinical studies.

Especially during the past two decades, there has been a growing awareness that exposure of individuals to exogenous environmental agents is responsible for various kinds of cancer. Chemical carcinogenesis in man was first documented in 1775 by the British physician Percival Pott, who attributed the high incidence of scrotal cancer in London chimney sweeps to their chronic exposure to soot and coal tars[l]. In the two hundred years following that initial observation, at least 1000 chemicals have been shown to induce cancer in a wide variety of tissues [2]. As our civilization has become industralized, our food, air, and water have undergone increased contamination with a number of cancer producing chemicals. It is currently estimated that approximately 75 to 85 percent of human cancer may be directly associated with exposure to these environmental carcinogens [3].

4-Ipomeanol (IPO) is the first agent to undergo preclinical development at the National Cancer Institute (NCI) based principally on a specific biochemical-biological rationale for clinical investigation as an antineoplastic agent targeted against lung cancer. This disease-specific development of IPO was initially stimulated by observations that the compound was activated by metabolism, preferentially within the mammalian lung, specifically within bronchiolar Clara cells, and that its predominant toxicity was to the lung in most species. IPO is inactive or only minimally active against most conventional antitumor test systems. However, some human lung cancer cell lines, as well as a variety of fresh human lung tumor biopsy specimens, have been shown to be capable of mediating the in situ biotransformation of IPO to a potentially cytotoxic intermediate. In this report, the biochemistry, metabolism, preclinical pharmacology, and toxicology of IPO are reviewed and the clinical development plans for this unique and challenging new agent are presented.

A fluorometric assay for the cytochrome P-450-dependent enzyme system, aryl hydrocarbon hydroxylase (AHH), was performed in mitogen-activated human lymphocytes from over 300 different humans and from 64 baboons. Results reveal: a) an average interindividual variation in AHH activity of approximately 0.25 (coefficient of variation); range of activities among humans and baboon subjects of approximately 40-fold; c) both genetic and environmental determinants of interindividual variation, and d) high AHH activity in humans associated with primary lung cancer. Confirmation of these results awaits the development of improved methods for phenotyping humans and for prospective cancer patient studies. DNA probes might be employed in future studies to determine specific mRNA content, and to search for DNA polymorphisms in and near the human cytochrome P-450 gene.

THE ENVIRONMENT AND THE LUNG 39$ achieved by prior culture for five days in DMEM + 0.5% FCS) and fibroblast cell counts determined after one, two, and four days. DMEM + 0.5% FCS ± asbestos incubated for four hours as controls. Supernatants from unexposed PAM had no influence on fibroblast replication rate. In contrast, supernatants from PAM exposed to asbestos significantly increased the rate of fibroblasts doubling; the doubling time decreasing five-fold from a control of 310±21 hours to 54±4 hours, P

Phagocytosis of asbestos bodies by human free alveolar macrophages (FAMs) was documented employing light microscopy. This process was more carefully studied utilizing scanning electron microscopy (SEM) which demonstrated morphological and surface membrane changes in FAMs following phagocytosis of asbestos bodies. FAM viability was also evaluated following 24--72-h incubation of cells with asbestos bodies at a final concentration of 250 micrograms/ml. Slight, but significant, cytotoxicity was observed following the initial 24-h culture period (P = 0.032, paired, 2-tailed t-test). No further cytotoxicity was observed, however, when cells were further incubated for 48-h and 72-h intervals (P greater than 0.05 in all instances). These studies demonstrate asbestos bodies are readily phagocytized by cultured FAMs, and are only slightly cytotoxic to these human lung cells.

Summary Aryl hydrocarbon hydroxylase (AHH) was induced in human pulmonary alveolar macrophages (PAMs) cultured in the presence of benzanthracene. Time and dose—response curves were established for in vitro induction of this enzyme system in PAMs. In addition, a positive correlation was noted between the level of AHH activity in freshly lavaged PAMs and the in vitro inducibility of the enzyme in these cells from either nonsmokers or cigarette smokers. Measurements of the inducibility of AHH in cultured human PAMs provide an experimental system suitable for studying the mechanisms responsible for the initiation of pulmonary carcinogenesis.

Both short-term and long-term xenograft models may be useful in conjunction with an in vitro based disease-oriented drug screening program. Shortterm models may be most valuable in making initial assessments of the potential in vitro drug screening leads for in vivo use. We have previously shown that a substantial number of such leads may be subject to metabolic inactivation (1) and that this may be associated with a lack of therapeutic activity and a relative lack of toxicity in vivo. Certainly, rapid excretion or other pharmacologic factors may also render compounds inactive in vivo. Short-term as says may be very useful for identifying such compounds and thus setting priorities for further testing of in vitro drug leads in more rigorous longer-term models. Rational application of these longer-term models with particular attention to modeling of in situ vascular barriers, tumor microenvironment, and the natural history of the target diseases may facilitate identification and development of new drugs with significant clinical activity against the common adult solid tumors.