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The present study examined whether benzodiazepine (BZ) intake alters performance and selected hormonal and metabolic variables during submaximal exercise. Seven triathletes completed two cycling trials at 85% maximum O2 uptake starting 3 h after an ingestion of either a placebo (PLA) of gelatin or BZ (1.5 mg lorazepam) and continuing until exhaustion, according to a double-blind randomized protocol. Blood samples were collected at rest; 5, 10, and 15 min; and exhaustion for dopamine (DA), norepinephrine (NE), epinephrine (Epi), adrenocorticotropic hormone (ACTH), cortisol (CORT), insulin (INS), free fatty acid, blood glucose, and lactate (La) determinations. Time of cycling was not significantly changed after BZ or PLA administration (22.9 +/- 2.5 vs. 23.5 +/- 3.8 min, respectively). A decrease in CORT and an increase in INS (P < 0.05) were observed with BZ before cycling. In comparison with rest, exercise resulted in a decrease in INS and an increase in all the other variables investigated (P < 0.001), but DA, NE, Epi, ACTH, CORT, La, and free fatty acid were significantly less elevated under BZ (P < 0.05). No change was found in glucose and INS levels between the two treatments at the end of the test. There was a strong correlation under both PLA and BZ conditions between DA, NE, Epi, and ACTH and also between Epi and La levels. From these data, BZ intake did appear to alter metabolism but did not influence performance during intense submaximal exercise.

A simple and sensitive high-performance liquid chromatographic method was developed to screen and determine amiloride (I) in human urine. The detection limit of the method is 0.12 micrograms/ml and the recovery of amiloride from urine was 80.4-85.5% at different concentrations. The coefficients of variation were less than 2.8 and 4.4% for intra- and inter-assays, respectively. Total urinary excretion of I in 24 h after oral administration of 5 mg or 15 mg of I ranged from 22.0 to 33.3% of the total dose for three different subjects. I could be detected in urine up to at least 44 h after a 5-mg dose and 72 h after a 15-mg dose. A gas chromatographic-mass spectrometric (GC-MS) confirmatory method was established based on the methanolysis of I to methyl 3,5-diamino-6-chloropyrazine-carboxylate (II). The di-N-trimethylsilyl derivative of II showed very good GC-MS properties and provided reliable structure information for confirmation analysis of I. This is the first time that a reliable GC-MS method has been reported for the detection of urinary I.

The effects of cysteine conjugates of styrene, e.g. S-1/2-(phenyl-hydroxyethyl) cysteine (PEC) and its N-acetyl derivative (NAPEC) on the transport of p-amino-hippurate (PAH) ion in plasma membranes were studied in vitro using isolated rat renal brush-border membrane (BBM) and basolateral membrane (BLM) vesicles. The uptake of PAH was significantly inhibited by both PEC and NAPEC in both the membrane vesicles, as verified by decrease of the membrane/medium concentration ratio of PAH as the concentration of either PEC or NAPEC in the medium increased. These results show that both PEC and NAPEC are capable of interfering with the accumulation of PAH (a model organic anion for renal tubular transport system) by both energy-independent and energy-dependent carrier-mediated transport processes. The inhibition of PAH uptake in BBM vesicles due to 10 mM PEC or NAPEC was found to be nearly competitive, almost similar to probenecid, whereas in BLM vesicles such inhibition was found to be partially noncompetitive, as verified by the double reciprocal plots. Both PEC and NAPEC showed dose-dependent inhibition of the specific activity of the marker enzyme in each membrane, e.g. gamma-glutamyl transferase in BBM and Na(+)-K(+)-ATPase in BLM vesicles. However, no such inhibition was noticed with probenecid. The in vitro pretreatment with probenecid prevented the inhibition of gamma-glutamyl transferase activity in BBM due to PEC or NAPEC, but such was not the case for the Na(+)-K(+)-ATPase activity in BLM. In conclusion, the data suggest that the transport of cysteine or N-acetylcysteine conjugates of styrene by renal proximal tubular cells across both the membrane vesicles accompanied by the inhibition of the membrane-specific enzymes may lead to cellular dysfunction and consequently to the initial development of their nephrotoxicity.

The effects of S-(2-chloroethyl)-DL-cysteine (CEC) (a potent nephrotoxin) on the transport of p-aminohippurate ion (PAH) in renal plasma membrane vesicles isolated from rat renal cortex were studied in vitro. The uptake of PAH was significantly reduced in a dose-dependent manner in both the brush border membrane (BBM) and basolateral membrane (BLM) vesicles. These results demonstrate that CEC is capable of interfering with the accumulation of PAH (a model organic anion for renal tubular transport system) by both energy-independent and energy-dependent carrier-mediated transport processes. Probenecid, a typical inhibitor of the organic anion transport system, showed the highest inhibition of PAH uptake in both the membranes vesicles. These data indirectly suggest that transport by renal tubular cells may result in the accumulation of CEC in renal cellular organelles eventually in toxic concentrations. Thus, CEC showed both dose- and time-dependent inhibition of the activities of gamma-glutamyl transferase (a BBM marker enzyme) and Na+, K(+)-ATPase (a BLM marker enzyme), while no such inhibition was noticed with probenecid. Pretreatment with probenecid prevented the inhibition of the gamma-glutamyl transferase activity due to CEC in BBM, but failed to do so for the Na+,K(+)-ATPase activity in BLM vesicles. Thus, the data suggest that the inhibition of the activities of these membrane-specific enzymes by CEC could lead to the initial development of its nephrotoxicity.

To evaluate the potential risk of pulmonary damage due to aerial spraying of the insecticide fenitrothion, rat lungs were examined under light and electron microscopy at 3, 7, 21, and 60 days after exposure. Rats were exposed by a "nose-only" apparatus for 1 hr to 2 or 500 mg/m3 of aerosolized fenitrothion (15%) mixed with solvent Cyclosol 63 (35%) and diluent oil 585 (50%). Aerosol size particles were monitored by a light scattering apparatus. Only minor modifications of lung alveolar tissues were observed after exposure to the high concentration. At 3 days, discrete foci of mild inflammation were detected, including interstitial edema, cellular infiltration, and increased number of alveolar macrophages. At 7 days, signs of irritation were diminished while at 21 and 60 days alveolar tissues were essentially normal. Exposure to lower concentration induced more limited changes at 3 days; no modifications were seen at later periods. It is concluded that a single exposure to this fenitrothion mixture at 500 mg/m3 presents no serious hazard of pulmonary toxicity.

Ultrastructural and cytochemical changes in peroxisomes of Type II alveolar cells were investigated in butylated hydroxytoluene (BHT)-administered mouse lungs. Male mice were given a single injection of BHT (400 mg/kg, ip) and sacrificed 1 to 7 days later. By means of tracheal infusion, lungs were fixed with a 2% glutaraldehyde or half-strength Karnovsky solution. Type I epithelium was selectively destroyed by BHT and was replaced by cuboidal Type II cells. Type II cells proliferated and some became squamous, extended their cytoplasm, and might differentiate into Type I cells (Hirai, Witschi, and Côté (1977) Exp. Mol. Pathol. 27, 295-308). Peroxisomes, Type II cell constituents, were clustered around and continuous with endoplasmic reticulum. The shape of the peroxisomes became indistinct after Type I cell injury by BHT. Also the density of the matrix was reduced in proportion to the reduction in the peroxidatic activity of catalase. These changes were accompanied by a decrease in the number of peroxisomes. New pinocytotic vesicles, one of the Type I cell characteristics, were generated at the apical and basal cell surfaces. Therefore, these cells had characteristics intermediate between Type I and Type II cells. These findings may indicate further evidence of the origin of Type I cells from Type II cells.

The administration of some ketonic or ketogenic compounds prior to a challenging dose of CCl4 potentiates the hepatic damage induced by this haloalkane. However, nothing is known about the recovery from the liver injury in these cases of chemically induced potentiation. To investigate this problem, we performed a temporal analysis of the hepatotoxic response of male Sprague-Dawley rats to CCl4 following a single pretreatment (p.o.) with: n-hexane, 2-hexanone, 2,5-hexanedione (15 mmol/kg in corn oil), isopropanol, acetone (33 and 34 mmol/kg in water, respectively); or the vehicle alone (10 ml/kg). They received, 18 h later, an i.p. injection of CCl4 (0.1, 0.75 or 1.0 ml/kg) and were killed 24-120 h later. Liver damage was assessed biochemically (ALT, OCT) and morphologically. A good correlation between biochemical and morphological results was observed. The ketonic or ketogenic compounds studied potentiated the liver injury produced by 0.1 ml/kg CCl4. Relative ranking orders regarding severity of maximal hepatic damage induced and time needed for complete recovery of liver injury were established; time of recovery was dependent on the maximal severity of the lesion, regardless of the potentiation. The results show that the temporal evolution of CCl4-induced liver injury is not markedly influenced by the administration of ketonic or ketogenic compounds as pretreatments, but rather depends on the severity of the maximal damage induced by the overall treatment.

The purpose of this study was to investigate whether the tissue distribution of chlordecone (CD) and mirex (M) might explain the difference in the potentiation of CHCl3 liver injury. Male Sprague-Dawley rats received either a single oral dose of CD or M (1, 2.5, 5, 10, 25, or 50 mg/kg) or three single daily doses of CD or M (0.5, 2, or 10 mg/kg). Eighteen hours following the last treatment, the animals were divided into two groups. The first group was killed and residues of CD or M in plasma, kidney, liver, and adipose tissue were measured by gas-liquid chromatography. The other group received CHCl3 (0.5 ml/kg, po) and was killed 24 hr later. Biochemical and histological indices of liver injury were evaluated. CD administered either singly or repetitively is more effective than M in potentiating the CHCl3-induced liver injury. This was exhibited by a higher increase in the plasma activities of the enzymes alanine aminotransferase and ornithine carbamoyl transferase and a greater alteration of the morphological pattern. There was a very good correlation between biochemical and histological indices. However, the severity of the liver injury did not parallel tissue concentrations. With M, a relatively poor potentiation of the liver damage was observed although adequate hepatic concentrations were present.

A semiquantitative morphologic procedure has been applied to chlordecone potentiation of CHCl3-induced liver injury in male Sprague-Dawley rats. The distance of the injured area from the terminal hepatic venule (THV) to the portal area was measured and the damaged cells were classified by type. The results were plotted graphically, along with elevations in plasma enzyme activities (GPT and OCT), to depict the pattern of damage. Chlordecone pretreatment enhanced the severity of the CHCl3-induced cellular changes and increased the number of cells affected. Dosages of 5 mg/kg of chlordecone did not potentiate CHCl3 toxicity, but higher dosages (10-50 mg/kg) enhanced the toxic response in a dose-dependent manner.

A variety of chemicals potentiate haloalkane-induced liver injury, but structure-activity relationships are not apparent. Recent studies have shown that one structural determinant, a carbonyl moiety, is common to several potentiating agents. Thus five ketonic chemicals (acetone, 2-butanone, methyl n-butylketone, 2,5-hexanedione, Kepone) and three chemicals that are metabolized to ketones (isopropranol, 2-butanol, n-hexane) potentiate the liver injury produced by one or more haloalkanes. Potentiation also has been observed when haloalkanes are administered to animals in a state of metabolic ketosis produced by alloxan-induced diabetes or by 1,3-butanediol administration. These observations are consistent with the hypothesis that administration or generation of ketonic substances increases the susceptibility of the liver to the toxic actions of haloalkanes.

After administration to mice of butylated hydroxytoluene, the pulmonary alveolar epithelium adopts a biphasic pattern of regenerative proliferation. This hitherto-unnoticed pattern of epithelial repair in the lung was revealed by the investigation of stereologic parameters. The earliest evidence of epithelial injury involved the type I pneumocytes, whose necrosis and disappearance from the septal surface was shown by a lowered surface density (SV). Proliferation of the type II pneumocytes ensued: the volume density (VV) rose above normal soon after the onset of necrosis, only to decrease as the cells slowly differentiated into intermediate and then type I pneumocytes. A second peak of type II pneumocytes appeared as the denuding of septa persisted. This twofold proliferation was also shown by the numerical density count (NV). Differentiation into an intermediate pneumocyte was itself documented by the raised VV and SV values. These observations of a biphasic mode of proliferation of type II pneumocytes raise the question of an unsuspected, persistent action of the toxic agent within pulmonary alveoli and serve to document the homeostasis of epithelial regeneration.

In this experiment we aimed at developping a method of ready evaluation of lung alveolar toxic damage using morphometric measurements. Swiss-Webster mice were treated with different doses of diquat (i.p.) and the left lung was fixed at 0, 3, and 5 days after treatment. The technique of morphometry of histological sections embedded in glycol methacrylate (GMA) was used to quantify possible modifications of alveolar cells. Quantitative evaluation includes: alveolar macrophages, polymorphonuclear cells, type II and intermediate pneumocytes. The results indicate a lung dose-dependent response, observed in both inflammatory and epithelial regeneration components of the alveolar primary reaction. This method could be used to readily evaluate the pulmonary toxic potential of any substance.

A single intraportal injection of 6-OH-DA (50mg/kg) in rats results in a functional hepatic sympathectomy 6 days following the injection. Laparotomy and mild abdominal exploration under pentobarbital anesthesia resulted in elevated plasma glucose levels within 15 minutes as a result of activation of the sympathetic nerves to the liver and adrenal gland discharge. The hyperglycemic response to trauma was reduced by fasting. The hyperglycemic response was examined in untreated rats, 6-OH-DA pretreated rats, bilaterally adrenalectomized rats, and a group which had received 6-OH-DA pretreatment as well as bilateral adrenalectomy. In non-fasted rats the presence of intact sympathetic nerves or intact adrenals was sufficient to produce the hyperglycemia. To prevent the response both nerves and adrenals must be deactivated. The hepatic nerves are primarily responsible for the early hyperglycemic response to trauma in fasted rats while in fed animals the adrenals and hepatic nerves play a more equal role.

A study was performed on the hepatic reaction of mice to acute intoxication with virotoxins (alaviroidin, viroisin, deoxoviroisin, viroidin, deoxoviroidin) and phalloidin, cyclic peptides isolated from Amanita virosa mushrooms. Purified fractions were administered intraperitoneally at various dosages to determine the LD50 which ranged from 1.0 to 5.3 mg/kg, with viroidin, phalloidin, and viroisin being the most potent. The virotoxins and phalloidin induced hemorrhagic necrosis of the liver. The development of hepatic lesions was followed by enhanced serum alanine aminotransferase (ALT) activity as well as by light and electron microscopic changes. In additional groups, bile ducts were cannulated and bile was collected for 2 hr after injection of the peptides (1 mg/kg) to determine their cholestatic potential. The earliest changes in hepatocytes were plasma membrane invagination and cytoplasmic vacuole formation. At later time periods, erythrocyte accumulation was evident in vacuoles and in the cytoplasm. The severity of hepatic damage, as judged by morphologic analysis, was correlated with serum ALT activity. Two of the peptides tested (viroisin and phalloidin) decreased bile flow by more than 50% over control values. Mild ultrastructural alterations in the bile canalicular pole of hepatocytes were observed during the development of cholestasis. Since virotoxins, like phalloidin, are bound to actin, it is possible that their affinity to cellular actin may be responsible for their hepatotoxicity.

There is now growing evidence that asbestos fibers could act in association with genotoxic compounds, either as cocarcinogens or promoters, in the process of carcinogenesis. The hepatocyte/UDS assay system has been taken to advantage to investigate the capacity of fibers to modulate the effects of genotoxic compounds on the cell, as we previously demonstrated the hepatocytes can engage in phagocytosis of chrysotile fibers. Measurement of UDS was performed by a biochemical procedure involving liquid scintillation counting (LSC) of a purified DNA fraction as well as by radioautography. Both LSC and radioautography revealed that chrysotile asbestos fibers UICC B at concentrations up to 100 micrograms/ml do not elicit UDS, whereas 2-acetylaminofluorene (2-AAF) at low concentrations (0.05-0.625 micrograms/ml) significantly induces it in parallel positive controls. In an attempt to test the cocarcinogen hypothesis, cultures of hepatocytes were simultaneously exposed for 20 h to 2-AAF (0.05 and 0.25 micrograms/ml) and asbestos fibers (1 and 10 micrograms/ml) given as simple mixtures. It was found that the 2-AAF-induced UDS activity was the same whether fibers were present or not. This was observed with both UDS evaluation procedures at all concentration combinations selected. An analysis of variance applied to the data collected from several experiments confirmed that there was no significant 2-AAF-fiber interaction. Our data suggest the absence of intrinsic genotoxic properties for chrysotile fibers. They also indicate that the modulation of the cellular response to genotoxic agents by asbestos fibers is not detected under our test conditions and may require longer-term exposures to be expressed.

Male weanling Sprague-Dawley rats were administered phenobarbital or diphenylhydantoin (phenytoin, dilantin, DPH), or a combination of the two drugs, while receiving a high calcium, low phosphorus vitamin D-deficient diet. A Line Test evaluation for calcification of the distal end of the radius was performed to determine the presence of rickets and the healing of the disease. After a period of 16 days, rickets had developed in a uniform manner in all groups regardless of drug treatment. Four I.U. of vitamin D3 were then administered to each animal and they were sacrificed either 3 or 9 days later. Three days after the administration of D3, bone calcification had started in all groups, with the greatest response being observed in the control group. Nine days after D3 administration, bone calcification was clearly impaired in PB and PB-DPH treated animals and slightly impaired in the DPH treated group when compared to controls. Weight gain was also impaired after PB and combined PB-DPH treatments. Based on these observations, it is suggested that anticonvulsant drugs, and in particular PB, inhibit, in a yet undetermined fashion, the normal calcification of the growth cartilage which accompanies a single physiological dose of cholecalciferol in the rat.

Cultures of rat hepatocytes have been used to study the interaction of chrysotile asbestos fibers with epithelial cells. The hepatocytes were incubated for 20 h with chrysotile asbestos (UICC B and a preparation of short fibers: 90% less than 2 micron) at concentrations of 1 or 10 micrograms/ml. Morphologic studies were then performed by means of transmission electron microscopy. Fibers were incorporated into plasma membrane invaginations, cytoplasmic vacuoles and phagolysosomes. These observations demonstrate that rat hepatocytes can engage in phagocytosis of chrysotile fibers. The results are compared to those previously obtained with mesothelial cells and tracheobronchial epithelium, exposed to asbestos in vitro. The advantages of the hepatocyte model to investigate the effects on cells of the association of asbestos fibers with genotoxic agents in relation to carcinogenesis are discussed.

In this study we applied linear integration morphometry to characterize the pulmonary alveolar reaction to toxic injury and to study possible relationships between the major tissue and cell compartments of alveolar tissue, normal and injured. Acute alveolar injury of mild and severe intensity was induced in Swiss-Webster mice by the ip administration of the chemicals diquat (4 mg/kg) and butylated hydroxytoluene (BHT; 400 mg/kg). Animals were sacrificed at Days 1 and 2 after diquat treatment and at Days 1, 3, and 5 after BHT treatment. Sampling and analysis of alveolar tissue were conducted at both levels of light and electron microscopy. Thickness distributions of arithmetic and reciprocal intercepts, as well as the arithmetic (tau) and harmonic (tau h) mean thicknesses, were established for the following alveolar compartments: septum, alveolo-capillary barrier (ACB), type I and total epithelia, capillary endothelium, and interstitium. A relative measure of the pulmonary diffusion capacity and the capillary load of alveolar septa were also determined. The parameters calculated from these thickness distributions, such as their slopes, percentages of thin intercepts, and tau/tau h ratios, proved very sensitive and useful in the detection and characterization of morphological alterations in the type I epithelial and capillary endothelial cells following either mild or severe alveolar injury. The epithelial, endothelial, and interstitial layers of pulmonary septa were all characterized by their own pattern of structural changes, so that it proved impossible to relate them in a simple way to the tissue reaction, which can be easily studied at the light microscopic level. Linear integration morphometry thus proved very useful as a morphometric approach to the study of pulmonary alveolar injury and repair.

The contribution of undifferentiated cells to callus formation is difficult to observe during fracture healing and is therefore debated. In adult mongrel dogs these cells can easily be observed in a narrow space separating a freshly inserted screw from the surrounding compact bone. Undifferentiated connective tissue cells invade these spaces from the medullary and, less so, from the periosteal site. They seem to resemble histologically and more so ultrastructurally B-type lymphocytes. They migrate to the site of repair or are brought there by the blood stream. Locally these cells proliferate and differentiated into osteoblasts. The authors prefer to call these cells lymphocyte-like cells.

Intraportal injection of 6-hydroxydopamine (25 mg/kg) results in nearly complete functional sympathectomy in the cat liver 1 week after the injection. Functional sympathectomy was verified by comparing the changes in liver blood volume (measured in vivo in a plethysmograph) resulting from direct stimulation of the hepatic nerves in control and pretreated animals. Pretreatment prevented the large reductions in hepatic blood volume normally seen with nerve stimulation while the response to infused noradrenaline was not reduced. Lack of morphological changes in right atrial nerve endings and an unaltered pressor response to bilateral carotid arterial occlusion indicate that sympathectomy at sites other than the liver did not occur.

Studies were performed to examine the effects of alloxan- or streptozotocin-induced diabetes on carbon tetrachloride (CCl4) liver injury. Male rats were pretreated with single i.v. injections of alloxan monohydrate (40 or 80 mg/kg) or streptozotocin (65 mg/kg). A challenging dose of CCl4 (0.1 ml/kg i.p.) was given to rats 4 days after alloxan pretreatment or 5 days after streptozotocin pretreatment, and the animals were sacrificed 24 hours later. Biochemical and morphologic evidence was obtained to show that pretreatment with the diabetogenic agents markedly enhanced CCl4-induced hepatotoxity. The challenging dose of CCl4 had no effect on the serum glutamic pyruvic transaminase (SGPT) activity in control rats. However, the administration of this dose of CCl4 to rats pretreated with 40 and 80 mg/kg of alloxan as well as to rats pretreated with streptozotocin resulted in 11-, 68-, and 32-fold increases, respectively, in SGPT activity. Hepatic triglyceride concentrations in the diabetic rats were also markedly elevated above control values after CCl4 challenge. Alloxan- or streptozotocin-pretreatment alone did not enhance these biochemical parameters of liver injury. Hepatic glucose-6-phosphatase activity, which increased in the rats given a diabetogenic agent, was lowered as a result of CCl4 injection. Insulin treatment of rats given alloxan (80 mg/kg) markedly protected against CCl4-induced hepatotoxicity. The severity of the morphologic changes in diabetic rats given CCl4 correlated with the biochemical findings.

Butylated hydroxytoluene has been previously shown to damage alveolar type-I cells followed by proliferation of type-II pneumocytes which then redifferentiate into intermediate and newly formed type-I epithelial cells. In relation to this transformation process, in vivo incorporation of H3-choline, a precursor of the main component of lung surfactant, was studied in type-II cells of BHT-treated mice by electron microscopic radioautography. The labeling density was reduced in some type-II cells at 2, 3 and 5 days after a single injection of BHT. Cellular compartment analysis indicated that the transformation of type-II cells is accompanied by a notable loss of their lamellar body component as well as a decreased radioautographic labeling associated with this cell type, possible in relation with modified synthesis of phospholipid material of surfactant.

The morphological heterogeneity of the lung and the poor extent of knowledge concerning alveolar repair following toxic insult have made morphology, and especially morphometry, a most suitable approach for the study of the injured lung. In this study we aimed to further develop a rapid and quantitative morphological approach to evaluate the sequence of lesion-repair in the aggressed alveolus. Swiss-Webster mice were treated with butylated hydroxytoluene (BHT, 400 mg/kg ip) and the lung was sampled at 1, 3, 5, 7, and 14 d after treatment. A morphometric evaluation, carried out on histological sections, was used to quantify the inflammatory and epithelial regenerative components of the alveolar primary reaction. Classical morphometric parameters such as the Aa and Na ratios, and the mean cellular surface were determined by planimetry. Following BHT administration, the alveolar stem cells (type II pneumocytes) proliferate and differentiate according to a biphasic pattern, with proliferative peaks at d 3 and 7. Furthermore, the challenged pulmonary alveolus retains increased numerical and surface density of macrophages and type II pneumocytes as late as d 14 after initial aggression.

Damage to the lung may be caused by chemicals that gain access to the alveolar zone by inhalation or via the pulmonary circulation. Several agents toxic to the lung have recently been found to bind covalently to pulmonary macromolecules or to disrupt certain metabolic reactions. However, it has also been observed that extensive chemical lung injury is not necessarily preceded by a depression of pulmonary metabolic reactions. One possible explanation for this might be that biochemical changes due to cell death are often masked and/or compensated for by changes associated with lung tissue repair. Substantial cell proliferation as a response to toxic lung damage is a common phenomenon in lung pathology. This makes it necessary to develop models that permit analysis of the biochemical events triggering and accompanying cell growth in lung. We have recently examined some aspects of cell proliferation in mouse lung. Intraperitoneal injection of the antioxidant butylated hydroxytoluene (BHT) produces within 3-5 days extensive hypertrophy, hyperplasia, and general disorganization of the cellular components of the lung. Total lung weight and total DNA per lung almost double within this time and are accompanied by proportional increases in protein and lipids. RNA accumulates at a faster rate than DNA. The changes in lung composition are accompanied by dose-dependent increases in the in vivo incorporation of thymidine into DNA and of leucine into protein. The activities of several enzymes (thymidine kinase, DNA polymerase, uridine kinase, glucose-6-phosphate dehydrogenase, and 5'-nucleotidase) increase substantially after BHT. Administration of BHT to mice seems to offer a convenient tool to study cell growth in the lungs of mice.

In this study we describe, through stereological methods, the lung morphology following inhalation exposure of guinea pigs to 21 mg/m3 cotton dust (CD) for 1 year. Various stereological parameters were determined on semithin histological sections, through a multistage sampling approach, to study the reaction of the whole lung, alveolar parenchyma, and bronchioles to CD inhalation. After 1 year of exposure, the lung volume was increased. Two distinct patterns of lung response were identified among the exposed animals. In type I responders, most of the morphometric parameters measured to describe the alveolar parenchymal reaction were within control value limits (means +/- 2 SD). In type II responders, the volume density (Vv) of the parenchymal zone was decreased, while the Vv, mean thickness, and absolute volume of the alveolar septa were increased. These changes caused the surface density (Sv) of alveolar epithelium to decrease, and an estimate of the percentage of alveolar septa remaining functional for gas exchange was also significantly lowered in these animals. In both types of responders, fifth to ninth orders of bronchioles had a raised wall to lumen ratio; the Vv and mean thickness of the bronchiolar epithelium were markedly increased, denoting hyperplastic changes. Thus, chronic exposure to cotton dust induced definite morphological changes on the peripheral conducting airways in most of the exposed animals, and induced pronounced changes at the alveolar level in 8 of 17 CD-exposed guinea pigs.

In this study we investigated the effects of a mild toxic challenge at selected points in time on the nonspecific cellular events that occurred in acutely damaged pulmonary alveoli. Swiss-Webster mice were treated with butylated hydroxytoluene (BHT, 400 mg/kg ip) and sacrificed at Days 1, 3, and 5 thereafter; either 24 or 48 hr prior to each sacrifice, the herbicide diquat was administered (4 mg/kg ip) as a challenge to the ongoing cellular events in the pulmonary alveoli. Standard morphometric techniques were used at both the levels of light and electron microscopy to evaluate the alveolar response to BHT and diquat treatments. Following BHT, early type I epithelial and endothelial damage triggered inflammatory changes in alveolar septa. Proliferation and differentiation of type II pneumocytes, aiming at the regeneration of the respiratory epithelium, ensued and peaked at Day 3. Treatment with diquat alone caused mild inflammatory changes and hypertrophy of type II pneumocytes, in the absence of necrosis of any alveolar cell type. The pinpoint administration of diquat in the early days following treatment with BHT significantly disorganized the temporal pattern of alveolar reaction. Diquat enhanced epithelial and endothelial damage only if administered before the onset of BHT-induced injury. Afterwards, the alveolar response to the combined effects of BHT and diquat could not be predicted from their known individual effects. Treatment with diquat modified either proliferation or differentiation of type II pneumocytes, depending upon time along the BHT schedule. Inflammatory and interstitial reactions were lowered when diquat was given at Days 1 and 3 post-BHT, but potentiated when given at Day 4. The results document time-related changes in the sensitivity of damaged and regenerating alveolar cells to a mild exogenous chemical challenge. They may further indicate that low levels of urban and industrial toxicants might influence pulmonary alveolar events in individuals made more susceptible by acute or chronic respiratory diseases.

The primary ultrastructural changes in pulmonary alveolar epithelial cells are described in paraquat-injected rats. Within 6-12 hr a single intravenous injection of 40 mg/kg paraquat dichloride caused selective mitochondrial swelling and loss of intramitochondrial granules within 24 hr in alveolar Type II cells. As the mitochondrial injury advanced, microvilli disappeared from apical plasma membrane followed by a cell destruction and detachment from basement membrane. This was accompanied by secondary damage to Type II cells and interstitial cells. These results indicate that paraquat may affect primarily the Type II cells and the first lesion produced occurs in mitochondria.

Intravenous injection of rats with 156 mumol/kg of paraquat or 140 mumol/kg of diquat produced, within 60 min, a sharp drop in the ratios of NADPH to NADP in lung. The effect persisted for a time period of at least 24 h. Exposure to 100% oxygen enhanced the toxicity of both compounds without substantially amplifying changes in the NADPH/NADP ratio. Lungs retained the capability to synthesize adenine nucleotides de novo. Electron microscopic studies showed that both paraquat and diquat damage type I alveolar cells, but only paraquat produces type II cell lesions. Although bipyridylium herbicides produce acute oxidation of NADPH in vivo, there seems not to exist a straightforward relationship between this event and cell damage.