Transfer of benzo(a)pyrene into mouse embryos and fetuses

The distribution of radioactive material within maternal and embryonic/fetal mouse tissue was studied over a period of 2 days following a single dose of¹⁴C-benzo (a)pyrene (B(a)P) orally on days 11, 12, 13 or 18 of pregnancy. B(a)P poorly penetrated into the embryo/fetus, and¹⁴C-radioactivity was found in embryonic tissues at concentrations one to two orders of magnitude lower than in maternal organs. If the compound was administered for 3 consecutive days at the same dose the expected cumulation was compensated for by an accelerated elimination of the substance, which is probably due to enzyme induction.     

Aryl hydrocarbon receptor-independent up-regulation of intracellular calcium concentration by environmental polycyclic aromatic hydrocarbons in human endothelial HMEC-1 cells

Polycyclic aromatic hydrocarbons (PAHs) such as benzo(a)pyrene (B(a)P) constitute a major family of widely‐distributed environmental toxic contaminants, known as potent ligands of the aryl hydrocarbon receptor (AhR). B(a)P has been recently shown to trigger an early and transient increase of intracellular calcium concentration ([Ca²⁺]_i), involved in AhR‐related up‐regulation of target genes by B(a)P. This study was designed to determine whether AhR may play a role in [Ca²⁺]_i induction provoked by B(a)P. We demonstrated that, in addition to B(a)P, various PAHs, including pyrene and benzo(e)pyrene, known to not or only very poorly interact with AhR, similarly up‐regulated [Ca²⁺]_i in human endothelial HMEC‐1 cells. Moreover, α‐naphthoflavone, a flavonoïd antagonist of AhR, was also able to induce [Ca²⁺]_i. Knocking‐down AhR expression in HMEC‐1 cells through transfection of siRNAs, was finally demonstrated to not prevent B(a)P‐mediated induction of [Ca²⁺]_i, whereas it efficiently counteracted B(a)P‐mediated induction of the referent AhR target gene cytochrome P‐450 1B1. Taken together, these data demonstrate that environmental PAHs trigger [Ca²⁺]_i induction in an AhR‐independent manner. © 2011 Wiley Periodicals, Inc. Environ Toxicol, 2012.     

Induction of hepatic mono-oxygenase systems of pregnant rats with phenobarbital and 3-methylcholanthrene.

When sodium phenobarbital was given to pregnant and non-pregnant female rats (40 mg/kg for 4 days), ethylmorphine N-demethylase, a cytochrome P-450-dependent system, was induced about 4-fold in non-pregnant females, but only 2-fold in pregnant females. The induction of microsomal cytochrome P-450 was also lower in pregnant animals. This impairment of phenobarbital induction occurred within 3 days of conception and disappeared after parturition within 5 days. 3-Methylcholan-threne induction of hepatic benzo[a]pyrene hydroxylase, a cytochrome P₁-450-dependent mono-oxy-genase system not inducible by phenobarbital, was not impaired during pregnancy. The depressed response of the maternal liver to phenobarbital induction can be partially reversed by the coadministra-tion of 3-methylcholanthrene. The administration of a higher dose of sodium phenobarbital (80 mg/kg day for 4 days) overcame the pregnancy-related lowered response to phenobarbital induction observed with the smaller dose of the barbiturate. The similarity in responses of the maternal and fetal livers to inducing agents suggests that a common regulatory mechanism operates in both the fetus and the pregnant female.     

Transformation of BHK 21/CL 13 cells by various polycyclic aromatic hydrocarbons using the method of styles

Nine polycyclic aromatic hydrocarbons (PAHs) were investigated by the cell-transformation assay method of Styles. Benzo(a)pyrene [B(a)P], chrysene (CH), 3-methylcholanthrene (3-MC), benz(a)anthracene (BA), benzo(b)fluoranthene [B(b)F], and dibenz(a,h)anthracene (DBA) were tested, including liver homogenate, and showed dose-effect relationships and a more than 2-fold increase of transformation rates at LC₅₀. Due to variations of the test method our results differed quantitatively from the data published by Purchase and Styles. Discrimination between the known carcinogens listed above and the noncarcinogens, phenanthrene (PA) and anthracene (AC), lacking a dose-effect relationship was, however, possible. Benzo(e)pyrene [B(e)P] was regarded as positive although producing only a 2-fold increase in the number of transformed colonies.     

蛇葡萄素与苯并芘合用对大鼠肝组织内CYP和GST基因表达的影响

目的观察蛇葡萄素与苯并芘合用对♂SD大鼠肝组织内CYP(cytochrome P450,CYP)和GST(glutathione S-transferase,GST)基因表达的影响。方法♂SD大鼠,蛇葡萄素(二氢杨梅素)250、500mg·kg-1,每日1次连续灌胃15d,d15模型组和2个给药组分别腹腔注射(ip)苯并芘100mg·kg-1。用逆转录-实时荧光定量PCR法检测肝组织内的CYP基因CYP1A1、CYP1A2、CYP1B1和GST基因GST-m1、GST-pi的表达情况。结果与空白组相比,苯并芘组(模型组)可明显诱导肝组织内的CYP1A1、CYP1A2、CYP1B1基因表达(P<0.01),分别是空白对照组的121倍、11倍、684倍,对谷胱甘肽-S转移酶基因GST-m1和GST-pi的表达有抑制趋势,分别是空白对照组的0.79倍和0.82倍;蛇葡萄素(二氢杨梅素)组+苯并芘组可明显诱导肝组织内的CYP1A1、CYP1A2、CYP1B1基因表达(P<0.01),分别是空白对照组的189和289倍、16.9和44.5倍、914和804倍,蛇葡萄素500mg.kg-1+苯并芘组明显诱导谷胱甘肽-S转移酶基因GST-m1和GST-pi的表达,分别是空白对照组的2.18倍(P<0.01)和2.12倍(P<0.05)。与苯并芘组(模型组)相比,蛇葡萄素250mg·kg-1+苯并芘组和蛇葡萄素500mg·kg-1+苯并芘组不影响CYP1B1的基因表达,但明显诱导CYP1A1的基因表达,分别是苯并芘组(模型组)的1.56倍(P>0.05)和2.39倍(P<0.05);明显诱导CYP1A2的基因表达,分别是苯并芘组(模型组)的1.53倍(P<0.05)和4.04倍(P<0.05);蛇葡萄素500mg.kg-1+苯并芘组明显诱导谷胱甘肽-S转移酶基因GST-m1和GST-pi的表达,分别是苯并芘组(模型组)的2.78倍(P<0.01)和2.57倍(P<0.05)。结论与苯并芘合用,蛇葡萄素(二氢杨梅素)可明显诱导CYP1A1、CYP1A2基因和谷胱甘肽-S转移酶基因GST-m1和GST-pi的表达。对CYP1B1基因无影响。表明蛇葡萄素与苯并芘合用可加速苯并芘代谢形成非致癌物而解毒和加速排泄。提示蛇葡萄素可对抗苯并芘的致癌作用。     

In vitro exposure to cigarette smoke induces oxidative stress in follicular cells of F1 hybrid mice

This study assessed the influence of cigarette smoke condensate (CSC) and benzo(a)pyrene [B(a)P] on the levels of two oxidative stress biomarkers [8‐isoprostane (8‐IsoP) and 8‐hydroxy‐2‐deoxy Guanosine (8‐OH‐dG)], in in‐vitro spent media of follicle cells. Follicles (100–130 µm) isolated from ovaries of F₁ hybrid (C57Bl/6j × CBA/Ca) mice were cultured for 13 days in media exposed to B(a)P [0 ng ml^–1 (control) to 45 ng ml^–1] or CSC [0 µg ml^–1 (control) to 130 µg ml^–1]. The concentrations of oxidative stress biomarkers in spent media were quantified by enzyme‐linked immune sorbent assays (ELISA). CSC and B(a)P treatment induced a significant, dose‐dependent increase in the concentrations of 8‐IsoP and 8‐OH‐dG in the spent media. We conclude that CSC and B(a)P exposure can induce oxidative stress in ovarian follicles, an effect that may contribute to the previously documented decline in follicle development and premature ovarian failure in women who smoke. © Her Majesty the Queen in Right of Canada 2013.     

Transplacental induction of mixed-function oxygenases in extra-hepatic tissues by 2,3,7,8-tetrachlorodibenzo-p-dioxin

Treatment of pregnant rats with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in a dose-dependent induction of a mixed-function oxidase system in fetal and maternal extra-hepatic tissues. At doses of 6 μmg/kg, aryl hydrocarbon hydroxylase (AHH) activity was increased 24-, 22- and 4-fold in fetal lung, kidney and skin, respectively, while maternal lung, kidney and adrenal AHH activity was increased 4-, 2- and 2-fold respectively. High-pressure liquid chromatographic (H.P.L.C.) analysis of benzo(a)pyrene (BP) metabolism after TCDD induction indicated that fetal lung, kidney and skin produced significant quantities of benzo(a)pyrene-7,8-dihydrodiol (BP-7,8-diol), benzo(a)pyrene-4,5-dihydrodiol (BP-4,5-diol) and 9- and 3-phenols of BP. The fetal liver produced benzo(a)pyrene-9,10-dihydrodiol (BP-9,10-diol), BP-4,5-diol, BP-7,8-diol and 9- and 3-phenols of BP. Maternal lung also produced BP-9,10-diol, while maternal adrenal gland yielded primarily the 9-phenol of BP. Epoxide hydratase activity was increased 2- to 3-fold in maternal lung, fetal lung and skin after TCDD pretreatment, but was not affected significantly in liver, kidney or placenta. Treatment of pregnant rats with TCDD increased the covalent binding of BP to DNA in preparations containing maternal liver, lung and placenta as well as fetal liver, lung and skin. Pretreatment with TCDD resulted in increased epoxide hydratase and AHH activities in extra-hepatic tissues but only AHH was increased in hepatic tissues, indicating that the inducing capabilities of TCDD differ from, but share some similarities with, both phenobarbital (PB) and 3-methylcholanthrene (MC). Thus, TCDD appears to provide an exceptionally potent and broad-spectrum transplacental induction of carcinogen-transforming enzymes in extra-hepatic tissues.     

Induction of aryl hydrocarbon hydroxylase in rat tissue following intratracheal instillation of diesel particulate extract and benzo[a]pyrene

The potential for aryl hydrocarbon hydroxylate (AHH) induction by inhaled diesel particles was assessed by intratracheal administration. Benzo[a]pyrene, (B[a]P) a reference compound, or an extract of particles, dissolved in gelatin-saline solution was administered to Fischer 344 rats at several dose levels. Twenty-four hours after administration of B[a]P or diesel particulate extract, the AHH activity increased in a dose-response manner in the lung, but not in liver. The maximum increase in the AHH activity in the lung was observed 12 h after intratracheal instillation of B[a]P (5 mg/kg), and the levels remained elevated for seven days. The AHH activity in the liver reached the maximum 24 h after the administration, and returned rapidly to control values. In contrast, intratracheal instillation of diesel particulate extract resulted in a significant increase of AHH activity in the lungs only after doses greater than 6 mg kg-1. The activity, however, declined rapidly and returned to control values within 75 h. The liver AHH activity in this instance, remained unchanged throughout the experimental period. These data indicate that in the lung, hydrocarbons extracted from diesel particles are weak enzyme inducers and exposure to these compounds by intratracheal administration did not result in AHH induction in the liver. The results suggest that doses higher than those normally expected from occupational exposures will be required to induce AHH activity in organs examined.     

Molecular dosimetry of DNA adducts in rainbow trout (Oncorhynchus mykiss) exposed to benzo(a)pyrene by different routes

 Farm raised rainbow trout (Oncorhynchus mykiss) were exposed by various routes to benzo(a)pyrene (BP) as a representative carcinogenic polycyclic aromatic hydrocarbon (PAH). Following exposure of fish to the chemical by intraperitoneal (i.p.) injection, ³²P-postlabelling studies indicated that non-feral trout were relatively resistant to the formation of BP-DNA adducts in liver. No adducts were detected in fish exposed to single doses (20 mg/kg) of BP. Multiple exposures (e.g. 2×25 mg/kg) were necessary in order for adducts to be detected, indicating that induction of the metabolising enzymes required for the bioactivation of BP is necessary. These studies provided reference information on DNA adducts for comparison with data from subsequent experiments at environmentally realistic low level exposures. Two types of low level aquatic exposure were carried out. The first procedure exposed fish for 30 days to a nominally constant low level (1.2 and 0.4 μg/l) of a homogeneous dispersion of BP in water, to simulate low level aquatic environmental exposures. Following ³²P-postlabelling analysis of the liver DNA of exposed fish, BP-DNA adducts were not detected. In the second procedure, fish were exposed to a constant low level of BP (ca. 0.5 μg/l) for 15 days then to a pulse (60 μg/l) which was allowed to naturally decline (to ca. 2 μg/l) during a further 15 days. Following this exposure, significant levels of BP-DNA adducts were detected in livers of trout. The effect of dietary exposures was investigated by feeding trout a diet containing either 58 μg or 288 μg BP per day for 6 days, equivalent to total doses of 43 mg/kg and 216 mg/kg. In both cases BP-DNA adducts were detected in livers of exposed fish. The results provide useful information on the types of exposures to PAHs which may pose a genotoxic risk to fish in the environment. Received: 5 April 1994 / Accepted: 31 May 1994     

Distribution to the fetus and major organs of the rat following inhalation exposure to pyrene.

Concentrations of pyrene and total metabolites were determined for individual fetuses and selected maternal organs and tissues immediately and 6 h following a 95-min head-only exposure of pregnant Wistar rats, on gestation day 17, to five levels of pyrene over the range 200-800 mg m-3 as a microcondensation aerosol. The influence of uterine horn, side and position, on distribution to the fetus was assessed. The concentration of both pyrene and its metabolites increased more over the exposure range (eightfold) than did those in the fetus. Concentrations of pyrene or its metabolites in fetal tissues were not found to be related to its position on the uterine horn. There was an unexplained and significant (P less than 0.01) higher concentration of pyrene in fetuses on the right side relative to the left side of the uterine horn for the animals killed immediately following exposure. A comparison of the levels in maternal tissues measured immediately following the exposure and 6 h later showed that there was some redistribution of pyrene and its metabolites to the fat tissues, i.e. levels in the fat increased over the 6 h period following the exposure. Levels in the other tissues diminished during this period. In general, concentrations of pyrene and its metabolites were lowest in the fetal tissues relative to those in the sampled maternal organs and tissues.     

Benzo(a)pyrene hydroxylase activity in the whole implantation site,decidua, placenta and fetal membranes of the rat

Summary Treatment of pregnant rats with 40 mg benzo(a)pyrene (BP) per kg body weight p.o. 24 h prior to sacrifice results in an induction of BP hydroxylase activity in the whole implantation site, the decidua, the placenta, and the fetal membranes. In control animals, no enzyme activity can be measured in these tissues with the exception of the fetal membranes which occasionally have a very low BP hydroxylase activity. In the BP-treated rats the enzyme activity is at least 3 to 6 times higher in the fetal membranes than in the placenta. The disappearance rate of BP hydroxylase activity was measured in the whole implantation site for 48 h after treatment with 40 mg BP kg body weight on day 8 of gestation. In the whole implantation site, the maximum enzyme induction is observed within 18 h while 48 h after BP-treatment, the enzyme activity is very low. The disappearance rate of BP hydroxylase activity was also measured for 48 h in the placenta on day 12 and on day 19 of gestation, respectively. In the placenta the height of enzyme induction remains almost unchanged within a 48 h interval. After BP-treatment of rats, morphological alterations can be observed in the decidua which are characterized by a moderate enlargement of the vesicles of the rough endoplasmic reticulum while this treatment does not effect the fine structure of the fetal membranes.This work was supported by a grant from the Deutsche Forschungsgemeinschaft given to the Sonderforschungsbereich 29, Embryonale Entwicklung und Differenzierung (Embryonal-Pharmakologie).Parts of this paper were presented at the Fifth International Congress on Pharmacology, San Francisco, July 1972.     

Genetic differences between C57BL/6 and DBA/2 mice in the inductions of UDP-glucuronyl transferases for 3-hydroxybenzo(a)pyrene,p-nitrophenol,and bilirubin by 3-methylcholanthrene

In C57BL/6 mice, aryl hydrocarbon hydroxylase (AHH) increased 1 day after treatment with 3-methylcholanthrene, and induction of UDP-glucuronyl transferases for 3-hydroxybenzo(a)pyrene, p-nitrophenol and bilirubin in the liver microsomes was observed 2 to 5 days later. In DBA/2 mice, neither AHH nor transferase activities were influenced by 3-methylcholanthrene. These results suggest that induction of activating and detoxicating enzymes is genetically linked.     

The effects of tricentric chromium(III) propionate complex supplementation on pregnancy outcome and maternal and foetal mineral status in rat

Chromium(III) is an essential element for carbohydrate and lipid metabolism, and various chemical forms of this element are widely used as dietary supplements. Of particular interest is [Cr₃O(O₂CCH₂CH₃)₆(H₂O)₃]⁺ cation (CrProp), that has been proposed as an alternative source of Cr. However, its safety has not been studied completely. In this study we investigated the effects of CrProp supplementation on pregnancy outcome and maternal and foetal mineral status in the rat. Female Wistar rats (n = 20, 14 weeks old) were mated with males and, after successful conception were fed either AIN-93G diet supplemented with CrProp (100 mg Cr/kg diet, equals to 7.2 mg Cr/kg body mass/day) or non-supplemented diet (0.27 mg Cr/kg diet, equals to 0.02 mg Cr/kg body mass/day) for 21 days. Dams were sacrificed on 21 day of gestation, and their foetuses were examined for adverse effects. Maternal and foetal organs were analysed for minerals contents (Fe, Cu, Zn, Cr) using the AAS-method. Supplemental Cr given did not affect pregnancy outcome, litter size, body and inner organ masses, maternal blood biochemical indices. No abnormalities in gross organ morphology of foetuses were detected. Supplemental CrProp increased maternal liver and kidney Cr levels by 177% and 455%, decreased liver Cu and Zn concentrations by 9% and 12%, increased foetal liver Zn by 181%, and decreased kidney Cu level by 34%.     

Biotransformation of benzo[a]pyrene and 7-ethoxyresorufin and heme-staining proteins in microsomes from human fetal liver and placenta

Microsomal proteins from human fetal livers and mid-gestational and term placentas were stained for heme in an attempt to detect multiple forms of cytochrome P-450. In fetal liver microsomes five protein bands staining for heme in the mol. wt region 46,000-60,000 were found. In the placentas two bands were seen in the region 46,000-52,000. Fetal liver and placental microsomes were assayed for metabolism of benzo[a]pyrene (B[a]P and 7-ethoxyresorufin (7-EOR). The B[a]P metabolites were separated using high performance liquid chromatography. Following incubations with fetal liver microsomes, in general only phenols were detectable, while after incubations with mid-gestational as well as term placentas from smoking women, the 9,10-, 4,5- and 7,8-dihydrodiols were also formed. No quinones were detected. Placental microsomes from non-smoking women did not catalyse the formation of B[a]P metabolites. The 7-EOR O-de-ethylase activity was in the same range (2-5 pmol/min x mg microsomal protein) in the fetal livers as in the mid-gestational placentas. The activities were somewhat higher in the placentas originating from smokers. No correlation between enzymatic activities in vitro and intensity of any specific protein band was observed for the fetal livers of placentas studied.     

Comparison of hepatic drug-metabolizing enzymes induced by 3-methylcholanthrene and phenobarbital between pre- and postnatal rats

Effects of 3-methylcholanthrene (3MC) and phenobarbital (PB) on the hepatic drug-metabolizing enzyme system in fetal liver of rats were investigated. Intraperitoneal administration of 3MC (25 mg/kg, 72 and 48 hr before death) to pregnant rats significantly increased hexobarbital (HB) and aminopyrine (AM)-metabolizing activities in fetuses on the 21st day of gestation to 148.0 and 150.6% of control fetuses, respectively. In contrast, HB and AM-metabolizing activities in 4-day-old neonates and mothers were decreased by administration of 3MC on the 21st day of gestation. Benzo[a]pyrene (BP)-metabolizing activity, NADPH-cytochrome c reductase activity, and cytochrome P-450 content in 3MC-treated fetuses were significantly increased to 2143.6, 137.6, and 323.8% of the control, respectively. Following 3MC administration, the maximum absorption of the cytochrome P-450-CO difference spectra in liver microsomes of fetuses was observed at 449-450 nm. The induction profile following 3MC administration in the fetal livers was different from that in the neonatal and the maternal livers. On the other hand, intraperitoneal administration of PB (60 mg/kg, 72, 48, and 24 hr before death) significantly increased HB, AM, and BP-metabolizing activities in fetal livers to 263.7, 231.0, and 151.2% of the respective controls. The profile induced by PB in the fetal livers was similar to that in maternal livers. These results suggest that HB and AM-metabolizing enzymes in fetal livers treated with 3MC or PB possess the capacity to be induced, and the responsiveness of the drug-metabolizing enzyme system to 3MC during the prenatal stage may differ from the postnatal stage.     

Effect of cytochrome P-450 inhibition on tetrahydrofuran-induced hepatocellular proliferation in female mice

The studies presented were designed to investigate the effects of cytochrome P450 inhibition on tetrahydrofuran-induced hepatocellular proliferation in female B6C3F₁ mice. Groups of female B6C3F₁ mice were exposed to dynamic atmospheres containing tetrahydrofuran (THF) concentrations of 0, 5,400 or 15,000 mg/m³ for 6 h per day, for 5 consecutive days. One-half of the animals in each THF exposure group were pretreated with the cytochrome P450 inhibitor 1-aminobenzotriazole (ABT) at 100 mg/kg (i.p.) 1 h before the start of each THF exposure period. Treatment with THF at 15,000 mg/m³ caused marked microsomal enzyme induction in the liver. The cytochrome P450 content was nearly doubled (+98%), pentoxyresorufin-O-depentylase (PROD) and ethoxyresorufin-O-deethylase (EROD) activities were increased by 600% and 160%, respectively. ABT pretreatment effectively blocked microsomal enzyme induction at 15,000 mg/m³. THF exposure had no effect on the subcellular morphology of hepatocytes, whereas ABT-pretreatment caused centrilobular fatty change. THF at 15,000 mg/m³ caused increased cell proliferation in zone 3 (central vein region) of the liver (according to Rappaport), as indicated by a significantly higher PCNA (Proliferating Cell Nuclear Antigen) labelling index, but there were no effects at 5,400 ppm. ABT pretreatment prior to THF exposure at 15,000 mg/m³ caused an exacerbated proliferative response of mouse liver, significantly higher PCNA labelling indices being observed in zones 2 (midzonal region) and 3. The exacerbated proliferative response of mouse liver under conditions of inhibited THF metabolism suggests that the mitogenic effects are related to prevailing THF tissue concentrations and not to the generation of THF oxidative metabolite(s).     

Thallium determinations in fetal tissues and maternal brain and kidney

The transfer of Tl⁺ cations through the placental barrier of pregnant mice and rats was studied by comparing the thallium concentrations in the maternal brain and kidney and those in fetal tissue at times of 10 min to 50 h after dosage of the animals with 10 mg/kg body wt. Tl₂SO₄. The quantitative determinations were performed with field desorption mass spectrometry after dilution of the homogenised tissue samples with enriched stable isotopes of thallium. The total sample quantity required for one complete assay is 1–3 μg, the analysis time for one determination about 30 min.     

Fetal translocation and metabolism of PAH obtained from coal fly ash given intratracheally to pregnant rats

Polycyclic aromatic hydrocarbons (PAH) were extracted from coal fly ash collected from the electrostatic precipitator of a thermal power plant. The PAH extract was given intratracheally daily to pregnant rats (2 mg/100 g body weight) on d 18 and 19 of gestation. In addition on d 19 of gestation rats were also given [3H]benzo[a]pyrene intratracheally. Rats were sacrificed on d 20 of gestation, and the distribution of [3H]benzo[a]pyrene radioactivity and PAH of coal fly ash was studied in maternal lung, liver, and placenta, as well as in the liver and lung of the fetus. The radioactivity of intratracheally given benzo[a]pyrene was found in liver (68%), placenta (4%), fetal lung (1.9%), and fetal liver (1.4%) of maternal lung. Intratracheally administered PAH of coal fly ash were translocated to maternal liver and placenta, as well as to the liver and lung of the fetus. PAH of coal fly ash were also metabolized to several minor and major metabolites by maternal lung, liver, and placenta, as well as by the maternal fetal liver and lung. Some of the PAH metabolites in lung and liver were common; however, the major metabolite of liver, M-16, was different from the major metabolite M-16 of lung. The major PAH metabolite of placenta, M-5, and fetal liver, F-12, were common PAH metabolites. M-2 and M-6 of the placenta and F-5 and F-10 of the fetal lung were also common.