Radiological-pathological correlation in diffuse idiopathic pulmonary neuroendocrine cell hyperplasia (DIPNECH): imaging and histopathology.

AIM: To review histologically confirmed diffuse idiopathic pulmonary neuroendocrine cell hyperplasia (DIPNECH) cases and carry out a detailed pathological-radiological correlation to see if computed tomography (CT) can be used to confidently identify DIPNECH. MATERIALS AND METHODS: Twenty-three histologically confirmed DIPNECH patients in the shared database of two NHS Trusts were reviewed. CT images were reviewed by two independent radiologists, each of them with >10 years of experience in thoracic imaging. All histological specimens were reviewed by a single pathologist with >25 years of experience. The diagnosis of DIPNECH was made according to the current World Health Organization (WHO) definition included in the WHO 2015 classification of pulmonary tumours. The results on histology were compared to the presence of nodules and air trapping on CT. Demographic information and, when available, molecular imaging studies and pulmonary function tests were also considered. RESULTS: There are prototypal clinical and radiological findings reflecting the presence of underlying histological DIPNECH: middle-aged women with multiple small and scattered nodules due to the clustering and proliferation of neuroendocrine cells. At least one larger, dominant, lung nodule reflecting a carcinoid tumour is very common and mosaic attenuation/air trapping is seen approximately in 50% of cases in inspiratory scans. Airflow obstruction is rarely associated with histological bronchial or peribronchial fibrosis, which suggests other mechanisms must be involved in its development. CONCLUSION: CT can be used to predict pathological DIPNECH in the appropriate clinical setting. It is important to consider DIPNECH to avoid overdiagnosis of more sinister conditions such as lung cancer or metastases.

Biotransformation of benzo[a]pyrene and other polycyclic aromatic hydrocarbons and heterocyclic analogs by several green algae and other algal species under gold and white light.

This laboratory has shown that the metabolism of benzo[a]pyrene (BaP), a carcinogenic polycyclic aromatic hydrocarbon (PAH), by a freshwater green alga, Selenastrum capricornutum, under gold light proceeds through a dioxygenase pathway with subsequent conjugation and excretion. This study was undertaken to determine: (1) the effects of different light sources on the enzymatic or photochemical processes involved in the biotransformation of BaP over a dose range of 5-1200 mg/l; (2) the phototoxicity of carcinogenic PAHs and mutagenic quinones to a green alga; (3) the ability of other algal systems to metabolize BaP. Cultures were exposed to different doses of BaP for 2 days at 23 degrees C under gold, white or UV-A fluorescent light on a diurnal cycle of 16 h light, 8 h dark. Under gold light, metabolites of BaP produced by Selenastrum capricornutum were the dihydrodiols of which the 11,12-dihydrodiol was the major metabolite. Under white light, at low doses, the major metabolite was the 9,10-dihydrodiol. With increasing dose, the ratio of dihydrodiols to quinones decreased to less than two. With increasing light energy output, from gold to white to UV-A in the PAH absorbing region, BaP quinone production increased. Of other carcinogenic PAHs studied, only 7H-dibenz[c,g]carbazole was as phototoxic as BaP while 7,12-dimethylbenz[a]anthracene, dibenz[a,j]acridine and non-carcinogenic PAHs, anthracene and pyrene, were not phototoxic. The 3,6-quinone of BaP was found to be highly phototoxic while quinones that included menadione, danthron, phenanthrene-quinone and hydroquinone were not. The data suggest that the phototoxicity of BaP is due to photochemical production of quinones; the 3,6-quinone of BaP is phototoxic and is probably the result of the production of short lived cyclic reactive intermediates by the interaction of light with the quinone. Lastly, only the green algae, Selenastrum capricornutum, Scenedesmus acutus and Ankistrodesmus braunii almost completely metabolized BaP to dihydrodiols. The green alga Chlamydomonas reinhardtii, the yellow alga Ochromonas malhamensis, the blue green algae Anabaena flosaquae and euglenoid Euglena gracilis did not metabolize BaP to any extent. The data indicate that algae are important in their ability to degrade PAHs but the degradation is dependent on the dose of light energy emitted and absorbed, the dose of PAHs to which the algae are exposed, the phototoxicity of PAHs and their metabolite(s) and the species and strain of algae involved. All of these factors will be important in assessing the degradation and detoxification pathways of recalcitrant PAHs by algae.

Influence of particle dose on the cytotoxicity of hamster and rat pulmonary alveolar macrophage in vitro.

Silica and ferric oxide are common industrial exposures. Studies have indicated that all commonly occurring forms of crystalline silica can cause fibrotic lung disease. There is evidence to indicate that crystalline silica is carcinogenic in humans who have not developed silicosis, while amorphous silica is not carcinogenic in humans. An important biological response to particles deposited deep in the lung is their engulfment by pulmonary alveolar macrophages (AM). To assess the role of AM in silica-induced lung disease, particle size distribution and surface area of crystalline, gelled, precipitated, and fumed silica, ferric oxide, and aluminum oxide were characterized; the cytotoxicity of the particles to hamster and rat AM in vitro was measured at 0.0-0.5 mg/1 x 10(6) cells at 24 and 48 h using dye exclusion procedures. The count medium diameter for aluminum oxide, ferric oxide, and amorphous silica was equal to or less than 0.38 microns, while for crystalline silica the value was 0.83 microns. The surface areas for the amorphous silicas and the aluminum oxide ranged from 253 to 125 m2/g with gelled silica having the highest value; the values for crystalline silica and ferric oxide were 4.3 and 10.8 m2/g, respectively. Crystalline silica (1.6%) was detected in the fumed silica, while none was detected in precipitated or gelled silica. With gelled silica, based on the dose of the particle, the viability of the hamster AM decreased to 27% at 0.05 mg and to zero at 0.1 mg at 24 h. At doses of 0.05 and 0.1 mg of crystalline, precipitated, or fumed silica, the percent viability decreased significantly to 76-67% and 51-42%, respectively, and to zero at 0.5 mg. Macrophages viable at 24 h decreased further at 48 h compared with the control culture. The ferric oxide and the aluminum oxide showed minimal to no changes in viability. Similar results for the particles were obtained with rat AM. The results indicate that precipitated and fumed amorphous silica tested at equivalent doses are equally as toxic to AM lavaged from two species of rodents as crystalline silica; gelled silica is more toxic than crystalline. Ferric oxide and aluminum oxide are noncytotoxic in this system. The results of this study indicate that the dose as well as the surface area and surface characterization are important determinants in the cytotoxicity of hamster and rat AM to these particles.

Comparative metabolism of 7H-dibenzo[c,g]carbazole and dibenz[a,j]acridine by mouse and rat liver microsomes.

The comparative metabolism of the carcinogenic pollutants 7H-dibenzo[c,g]-carbazole (DBC) and dibenz[a,j]acridine (DBA) was investigated in vitro using 3-methylcholanthrene (3MC) induced Sprague-Dawley rat and Hsd:ICR(Br) mouse liver microsomal preparations with benzo[a]pyrene (BaP) as the positive control. Metabolites were isolated and separated by HPLC and identified by spectroscopic and co-chromatographic techniques using synthetic standards. The major metabolites of DBC were the phenols: the 5-OH-DBC, 3-OH-DBC, and 2-OH-DBC. Traces of 1-OH-DBC were also found yet no dihydrodiols were identified. The major metabolites of DBA were the 3,4-diol-DBA and 5,6-diol-DBA, 1,2-diol-DBA, DBA-5,6-oxide and 4-OH-DBA. Treatment of both mice and rats with 3MC resulted in significant (P less than or equal to 0.05) increases relative to control in the microsomal metabolism of DBA to dihydrodiol and phenol metabolites, similar to that observed for BaP. 3MC-induced rat liver microsomes significantly (P less than or equal to 0.05) increased DBC metabolism relative to control microsomes whereas DBC metabolism was not increased with 3MC-induced mouse liver microsomes. These data indicate that different enzymatic pathways are involved in the metabolic activation of DBC in the Hsd:ICR(Br) mouse and Sprague-Dawley rat.

Conjugation of benzo[a]pyrene metabolites by freshwater green alga Selenastrum capricornutum.

Benzo[a]pyrene (BaP) undergoes metabolic transformation in mammals via oxidative, hydrolytic, and conjugative processes; however, little is known concerning BaP conjugation in freshwater algae. It has been shown in this laboratory that BaP is metabolized by Selenastrum capricornutum via a dioxygenase pathway. This study describes the conjugation of BaP metabolites by a green alga, Selenastrum capricornutum. Cultures were exposed to 1160 micrograms/l [14C]BaP for 4 days at 23 degrees C under gold fluorescent lights on a diurnal cycle of 16 h light, 8 h dark. Of the total metabolites in the algal culture, 89% were present in media. BaP and non-conjugated metabolites were separated from conjugated metabolites by chromatography on neutral alumina columns using solvents of increasing polarity. Seventy-one percent of the BaP metabolites were conjugates of which 12.2%, 12.0% and 12.4% were sulfate ester and alpha- and beta-glucose conjugates, respectively. Conjugates that coeluted with sulfate esters were hydrolyzed with arylsulfatase, alpha- or beta-glucosidase; high performance liquid chromatography (HPLC) analysis indicated that the major product of each enzymatic hydrolysis was the 4,5-dihydrodiol (87.2, 69 and 53%, respectively). Eighty-six percent of the conjugates were acid labile following incubation for 2 h in 4 N HCl at 37 degrees C. To our knowledge this is the first demonstration of the metabolism of a polynuclear aromatic hydrocarbon by a freshwater green alga through a dioxygenase pathway and subsequent conjugation and excretion.

Metabolism of benzo(a)pyrene by a dioxygenase enzyme system of the freshwater green alga Selenastrum capricornutum.

The green alga Selenastrum capricornutum was incubated with benzo(a)-pyrene under an atmosphere of 20% (18)O2: 80% N2. The cis-11,12-dihydro-11,12-dihydroxybenzo(a)pyrene, cis-7,8-dihydro-7,8-dihydroxybenzo(a)pyrene and cis-4,5-dihydro-4,5-dihydroxybenzo(a)pyrene, were isolated by HPLC and analyzed by mass spectrometry. The metabolites produced molecular ions at m/z 290 and 286. Elemental analysis of the ion at m/z 290 gave an elemental composition of C20H14(18)O2 with 13% (18)O2 incorporation. The results indicate that S. capricornutum produces cis vicinal dihydrodiols from molecular oxygen via a dioxygenase enzyme pathway. The dioxygenase enzymes are characteristic of the bacterial metabolic pathway and unlike those of eukaryotic organisms which involve monooxygenase enzymes.

Metabolism of mutagenic polycyclic aromatic hydrocarbons by photosynthetic algal species.

Polycyclic aromatic hydrocarbons (PAH) known to produce carcinogenic and mutagenic effects have been shown to contaminate waters, sediments and soils. While it is accepted that metabolites of these compounds are responsible for most of their biological effects in mammals, their metabolism, and to a large extent their bioactivity, in aquatic plants have not been explored. Cultures of photosynthetic algal species were assayed for their ability to metabolize benzo[a]pyrene (BaP), a carcinogenic PAH under conditions which either permitted (white light) or disallowed (gold light) photooxidation of the compound. Growth of Selenastrum capricornutum, a fresh-water green alga, was completely inhibited when incubated in white light with 160 micrograms BaP/l medium. By contrast concentrations at the upper limit of BaP solubility in aqueous medium had no effect on algal growth when gold light was used. BaP quinones and phenol derivatives were found to inhibit growth of Selenastrum under white light incubation. BaP phototoxicity and metabolism were observed to be species-specific. All 3 tested species of the order Chlorococcales were growth-inhibited by BaP in white light whereas neither the green alga Chlamydomonas reinhardtii nor a blue-green, a yellow-green or an euglenoid alga responded in this fashion. Assays of radiolabeled BaP metabolism in Selenastrum showed that the majority of radioactivity associated with BaP was found in media as opposed to algal cell pellets, that the extent of metabolism was BaP concentration dependent, and that the proportion of various metabolites detected was a function of the light source. After gold light incubation, BaP diols predominated while after white light treatment at equal BaP concentrations, the 3,6-quinone was found in the highest concentration. Extracted material from algal cell pellets and from media was tested for mutagenicity in a forward mutation suspension assay in Salmonella typhimurium using resistance to 8-azaguanine for selection. Direct-acting mutagens were detected in extracted media from incubation of Selenastrum with 400 micrograms BaP/l for 1 day in gold light. Extracts of media from algae incubated in gold light from 1 to 4 days with 1200 micrograms BaP/l were found to have direct-acting mutagens as well as those requiring further metabolism. Media extracts from white light incubations of BaP were mutagenic upon addition of rat liver homogenates. Activity of these materials from white light treatment are largely attributable to unmetabolized BaP.

Intracisternal protein in the type II pneumocyte of the ferret, guinea pig, and mongrel dog.

An intracisternal protein in the type II pneumocyte of the ferret, guinea pig, and mongrel dog was examined by light and electron microscopy and morphometry. The basic pattern of layering in this membrane-bound, ribosome-studded structure (cisternal body) was visualized in cross section as dense layers separated by approximately 0.1 micron with seven fine layers between. In all species the central fine band of the seven was occasionally more prominent than the other six. In the guinea pig the seven fine layers alternated in density from light to dark. The cisternal body of the dog was similar to that of the ferret, but was very much smaller and encountered infrequently. No function has been ascribed to this structure; however, its relation to lamellar bodies, the perinuclear membrane, and surfactant apoprotein is discussed.

Mutagenicity of algal metabolites of benzo(a)pyrene for Salmonella typhimurium.

The metabolism and growth effects of benzo(a)pyrene (BaP) were studied using a freshwater green alga, Selenastrum capricornutum. Algal cultures were incubated under gold light with BaP added at concentrations of 40, 160, 400, and 1,200 micrograms/liter for the periods of 1-4 days. The metabolites and BaP were identified and quantified from ethyl acetate extracts of both algal cells and incubation medium. The ethyl acetate extracts were evaluated for genotoxicity using a micro-volume Salmonella typhimurium forward mutation assay with resistance to 8-azaguanine for selection. This assay detected the presence of small quantities of BaP and was particularly sensitive to the mutagenicity of BaP diols. Of those extracts prepared from algae and medium from cultures exposed to 400 micrograms BaP/liter (10 micrograms/25 ml culture), only algal cell extracts from one day's growth were mutagenic. In cultures exposed to 1,200 micrograms BaP/liter (30 micrograms/25 ml culture), mutagenic materials were produced or persisted in both algae and media throughout the 4-day incubation. The observed mutagenic response can be attributed in part to the presence of unmetabolized BaP or to BaP diols.

The phototoxicity of benzo[a]pyrene in the green alga Selenastrum capricornutum.

The effects of selected polycyclic aromatic hydrocarbons (PAHs) on the growth of the green alga Selenastrum capricornutum in three light regimens were examined. In gold fluorescent light, benzo[a]pyrene (BaP) at 12 mg/liter (48 mumole/liter), benz[a]anthracene (BaA) at 40 mg/liter (175 mumole/liter), anthracene (A) at 40 mg/liter (224 mumole/liter), and 13 metabolites of BaP each at 40 micrograms/liter had no effect on algal growth. In cool-white fluorescent light, 30% inhibition of algal growth occurred with 0.1 mumole/liter BaP, 8.0 mumole/liter BaA, and 40 mumole/liter A. BaP at 0.16 mg/liter (0.64 mumole/liter) totally inhibited growth. BaP concentrations an order of magnitude lower inhibited algal growth in fluorescent blacklight. In cool-white light, 5 of 13 metabolites of BaP (each 40 micrograms/liter) inhibited algal growth: 3,6-quinone; 6-hydroxy; 9-hydroxy; 3-hydroxy; and 1,6-quinone. Based on these results, PAHs and metabolites of BaP are selectively phototoxic to S. capricornutum due to the incident light intensity below 550 nm.

Effect of ethanol on vinyl chloride carcinogenesis.

Four treatment groups (80 male Sprague-Dawley rats/group) were used in a 2 X 2 factorial design: inhalation of 600 ppm vinyl chloride (VC) 4 hr/day, 5 days/week for 1 year; VC and ingestion of 5% ethanol in water (v/v); filtered air and ethanol; filtered air. Ingestion of ethanol was begun 4 weeks prior to inhalation of VC and continued for life or termination of the study at two and one-half years from the first VC exposure. In this model system, ethanol potentiated the carcinogenic response to VC in the liver and produced an excess of neoplasms in animals receiving ethanol alone. Inhalation of VC induced angiosarcoma of the liver in 23% of the exposed animals; ethanol in addition to VC inhalation increased the incidence to 50%. Concomitant administration of VC and ethanol also produced an excess of hepatocellular carcinoma and lymphosarcoma. Ethanol with or without VC had a strong tumorigenic effect on the endocrine system. These results indicate that ethanol is a cocarcinogen in relation to the carcinogen VC.

Effect of ethanol and vinyl chloride on the induction of liver tumors: preliminary report.

Preliminary results of a long-term animal study (rats) indicate synergism between inhaled vinyl chloride and ingested ethanol in tumorigenesis.