Early life exposure to allergen and ozone results in altered development in adolescent rhesus macaque lungs.

In rhesus macaques, previous studies have shown that episodic exposure to allergen alone or combined with ozone inhalation during the first 6 months of life results in a condition with many of the hallmarks of asthma. This exposure regimen results in altered development of the distal airways and parenchyma (Avdalovic et al., 2012). We hypothesized that the observed alterations in the lung parenchyma would be permanent following a long-term recovery in filtered air (FA) housing. Forty-eight infant rhesus macaques (30 days old) sensitized to house dust mite (HDM) were treated with two week cycles of FA, house dust mite allergen (HDMA), ozone (O3) or HDMA/ozone (HDMA+O3) for five months. At the end of the five months, six animals from each group were necropsied. The other six animals in each group were allowed to recover in FA for 30 more months at which time they were necropsied. Design-based stereology was used to estimate volumes of lung components, number of alveoli, size of alveoli, distribution of alveolar volumes, interalveolar capillary density. After 30 months of recovery, monkeys exposed to HDMA, in either group, had significantly more alveoli than filtered air. These alveoli also had higher capillary densities as compared with FA controls. These results indicate that early life exposure to HDMA alone or HDMA+O3 alters the development process in the lung alveoli.

Lung effects of inhaled corticosteroids in a rhesus monkey model of childhood asthma.

BACKGROUND: The risks for infants and young children receiving inhaled corticosteroid (ICS) therapy are largely unknown. Recent clinical studies indicate that ICS therapy in pre-school children with symptoms of asthma result in decreased symptoms without influencing the clinical disease course, but potentially affect postnatal growth and development. The current study employs a primate experimental model to identify the risks posed by ICS therapy. OBJECTIVE: To (1) establish whether ICS therapy in developing primate lungs reverses pulmonary pathobiology associated with allergic airway disease (AAD) and (2) define the impact of ICS on postnatal lung growth and development in primates. METHODS: Infant rhesus monkeys were exposed, from 1 through 6 months, to filtered air (FA) with house dust mite allergen and ozone using a protocol that produces AAD (AAD monkeys), or to FA alone (Control monkeys). From three through 6 months, the monkeys were treated daily with ICS (budesonide) or saline. RESULTS: Several AAD manifestations (airflow restrictions, lavage eosinophilia, basement membrane zone thickening, epithelial mucin composition) were reduced with ICS treatment, without adverse effects on body growth or adrenal function; however, airway branching abnormalities and intraepithelial innervation were not reduced. In addition, several indicators of postnatal lung growth and differentiation: vital capacity, inspiratory capacity, compliance, non-parenchymal lung volume and alveolarization, were increased in both AAD and Control monkeys that received ICS treatment. CONCLUSIONS AND CLINICAL RELEVANCE: Incomplete prevention of pathobiological changes in the airways and disruption of postnatal growth and differentiation of airways and lung parenchyma in response to ICS pose risks for developing primate lungs. These responses also represent two mechanisms that could compromise ICS therapy's ability to alter clinical disease course in young children.

Carcinogenicity of dietary aflatoxin M1 in male Fischer rats compared to aflatoxin B1.

Aflatoxin M1 (AFM), an hydroxy metabolite of the potent carcinogenic mycotoxin aflatoxin B1 (AFB) is frequently found in milk and other dairy products. Sufficient amounts of AFM were produced to study the carcinogenicity of this compound. AFM was fed to male Fischer rats starting at 7 weeks up to 21 months of age. Agar-based semisynthetic diets contained 0.0, 0.5, 5.0, and 50.0 micrograms/kg of AFM or 50 micrograms/kg of AFB. Hepatocellular carcinomas were detected in two of 37 rats and neoplastic nodules were found in six of 37 rats fed 50 micrograms/kg AFM between 19 and 21 months. No nodules or carcinomas were observed in the lower AFM dose groups. Nineteen of 20 rats fed a diet containing 50 micrograms/kg of AFB developed hepatocellular carcinomas by 19 months of age. Carcinogenic potency of the aflatoxins was reflected by morphometric quantitation of foci detected in hematoxylin and eosin stained sections. Three rats fed the diet containing 50 micrograms/kg AFM developed intestinal carcinomas. None were observed in other groups. Under the conditions of this experiment AFM was found to be a weak hepatic carcinogen compared to AFB and to possess intestinal carcinogenicity.

Trafficking of neutrophils across airway epithelium is dependent upon both thioredoxin- and pertussis toxin-sensitive signaling mechanisms.

Leukocyte recruitment from the circulation into the airways is a multi-step process, involving both chemotactic and adhesive mechanisms. Using an in vitro model of leukocyte transepithelial trafficking, we show that movement of human peripheral blood neutrophils (PMN) across airway epithelium in the optimal basolateral-to-apical surface direction is partially blocked by pertussis toxin, an inhibitor of G(alphai)-protein-linked receptors. A neutralizing monoclonal antibody against interleukin-8 (IL-8; constitutively expressed by airway epithelium) did not inhibit PMN transepithelial migration, suggesting that alternative pertussis toxin-sensitive signaling mechanisms are involved in this process. However, a neutralizing antibody against thioredoxin, a redox enzyme with pertussis toxin-insensitive chemoattractant activity, did reduce PMN migration across airway epithelium. We conclude that trafficking of PMN across airway epithelium is mediated by both thioredoxin- and pertussis toxin-sensitive signaling mechanisms that are independent of IL-8.

Substance P primes the formation of hydrogen peroxide and nitric oxide in human neutrophils.

Substance P (SP), a neurotransmitter of the central and peripheral nervous system, has been implicated as a mediator of the pulmonary inflammatory response through its stimulatory effects on neutrophils. We investigated the role of SP in priming the production of reactive oxygen species by human neutrophils with the cytochrome c reduction assay and by flow cytometry using the intracellular oxidizable probe dichlorofluorescein. We also investigated SP-induced formation of nitrite and nitrate as an index of nitric oxide (NO) production. Our results indicate that SP primes two distinct pathways with respect to the induction of reactive oxygen species in the human neutrophil: the production of superoxide anion and hydrogen peroxide by the calmodulin-dependent NADPH oxidase, and the generation of NO by a constitutive NO synthase. Preincubation of neutrophils with inhibitors of calmodulin and NO synthase diminished the oxidative response in an additive fashion. These results give insight into distinct signal transduction pathways in the SP-primed neutrophil with respect to the formation of superoxide anion, hydrogen peroxide, and NO.

Expression of the beta6 integrin subunit is associated with sites of neutrophil influx in lung epithelium.

Inhalation of ozone by Rhesus monkeys results in epithelial injury and granulocyte influx in both conducting airways and respiratory bronchioles. We have reported that ozone-induced neutrophil recruitment and subsequent epithelial repair can be inhibited in vivo with a CD18 antibody. The antibody-mediated effect is abrogated by local instillation of C5a (a CD18-independent neutrophil chemoattractant), thereby demonstrating a role for neutrophils in lung epithelial repair processes. As an extension of this study, we examined the effect of ozone and neutrophil influx on epithelial expression of the beta6 integrin, an adhesion molecule associated with proliferation and repair. Expression of beta6 integrin was determined by immunohistochemistry for ozone-exposed monkeys treated with either control immunoglobulins or a CD18 antibody. The tracheal epithelium of ozone-exposed monkeys treated with control immunglobulins expressed the beta6 integrin. In contrast, the tracheal epithelium of ozone-exposed monkeys treated with CD18 antibody exhibited very low to undetectable expression of beta6 integrin. In association with C5a instillation and neutrophil influx, beta6 integrin was also observed in respiratory bronchiolar epithelium from both control and ozone-exposed animals. These findings cumulatively suggest that lung epithelial cell expression of beta6 integrin is associated with sites of neutrophil recruitment.

Regional differences in quantities of histochemically detectable mucosubstances in nasal, paranasal, and nasopharyngeal epithelium of the bonnet monkey.

Inhaled irritants induce secretory cell hyperplasia in nasal epithelium of animals. To characterize this response histochemically it is first important to know the histochemical character and distribution of epithelial mucosubstance in the normal nasal cavity. An automated image analyzing method was used to detect and quantitate acidic, neutral, and sulfated mucosubstances in the epithelium lining the nasal and paranasal airways of eight bonnet monkeys. Tissue sections 2 micron thick from defined regions of these airways were stained with either alcian blue/periodic acid-Schiff to demonstrate acid and neutral mucosubstances or high iron diamine to demonstrate sulfated mucins. Respiratory epithelium covering maxilloturbinates had the largest volume of stainable mucosubstance per unit surface area of basal lamina, whereas the maxillary sinus epithelium had the least. There was a general anteroposterior increase in the quantity of total epithelial mucosubstance along the septal and lateral walls of the nasal cavity, and there was more acidic than neutral mucosubstance in the posterior nasal airway than in the anterior. Epithelial mucosubstance in the maxillary sinus was predominantly neutral. Therefore, we conclude that there are substantial regional quantitative differences in stainable mucosubstances in the primate nasal epithelium which must be considered when examining nasal mucosa for irritant-induced changes in epithelial mucins.

Quantitative histochemistry of mucosubstance in tracheal epithelium of the macaque monkey.

Experimentally applied irritants and chronic respiratory diseases appear to alter the amount and composition of secretory cell product in surface epithelium and submucosal glands of pulmonary airways. Previous methods used to quantify these changes have been very time-consuming or have not measured the same components of the airway wall. The present study describes a rapid, reproducible, and standardized automated method for quantifying secretory products. The tracheas from eight macaque monkeys were fixed with glutaraldehyde-paraformaldehyde, embedded in glycol methacrylate, serially sectioned at 2 microns, and histochemically stained to demonstrate neutral, sialylated, and sulfated mucosubstances in the cartilaginous, intercartilaginous, and membranous regions of both proximal and distal trachea. Volume densities were determined using an image analyzer and are expressed as volume of stained mucosubstance per unit surface area of epithelial basal lamina. Comparison of the automated method to manual point counting and evaluation of internal variance showed that the automated method had a twelve-fold increase in efficiency with no significant differences in measurements. After weighting the values of each region according to their anatomical contribution, the total secretory product (TSP) for the entire trachea was determined. Periodate-reactive acid material predominated (73%) in luminal surface epithelium, and neutral material predominated (78%) in submucosal glands. Surface epithelium contained 66% of the TSP. The greater contribution by surface epithelium and predominance of acid mucins there resulted in a TSP from the trachea that consisted of 59% acid material (most of which was sulfated) and 41% neutral material. The method proved to be a valid, reproducible, and rapid technique for evaluating variability in abundance of mucosubstances within airway epithelium.

Amelioration of bleomycin-induced pulmonary fibrosis in hamsters by combined treatment with taurine and niacin.

Interstitial pulmonary fibrosis induced by bleomycin (BL) involves the production of reactive oxygen species and the impairment of repair of damaged epithelial cells. We have shown previously that taurine or niacin treatment partially attenuates BL-induced lung fibrosis in hamsters and that the two agents probably act through different mechanisms. In the present investigation, we have demonstrated that taurine and niacin in combination provide nearly complete protection against BL-induced pulmonary fibrosis. Based on the findings of this investigation, it is suggested that combined treatment with taurine and niacin offers the potential for a novel pharmacological approach in the prevention of lung fibrosis in humans.

Antifibrotic effect of decorin in a bleomycin hamster model of lung fibrosis.

We reported previously that treatment with antibody to transforming growth factor-beta (TGF-beta) caused a marked attenuation of bleomycin (BL)-induced lung fibrosis (LF) in mice. Decorin (DC), a proteoglycan, binds TGF-beta and thereby down-regulates all of its biological activities. In the present study, we evaluated the antifibrotic potential of DC in a three-dose BL-hamster model of lung fibrosis. Hamsters were placed in the following groups: (1) saline (SA) + phosphate-buffered saline (PBS) (SA + PBS); (2) SA + DC; (3) BL + PBS; and (4) BL + DC. Under pentobarbital anesthesia, SA (4 mL/kg) or BL was instilled intratracheally in three consecutive doses (2.5, 2.0, 1.5 units/kg/4 mL) at weekly intervals. DC (1 mg/mL) or PBS was instilled intratracheally in 0.4 mL/hamster on days 3 and 5 following instillation of each dose of SA or BL. In week 4, hamsters received three doses of either DC or PBS every other day. The hamsters were killed at 30 days following the first instillation, and their lungs were appropriately processed. Lung hydroxyproline levels in SA + PBS, SA + DC, BL + PBS, and BL + DC groups were 965, 829, 1854, and 1387 microg/lung, respectively. Prolyl hydroxylase activities were 103, 289, and 193% of SA + PBS control in SA + DC, BL + PBS, and BL + DC groups, respectively. The myeloperoxidase activities in the corresponding groups were 222, 890, and 274% of control (0.525 units/lung). Intratracheal instillation of BL caused significant increases in these biochemical markers, and instillation of DC diminished these increases in the BL + DC group. DC treatment also caused a significant reduction in the infiltration of neutrophils in the bronchoalveolar lavage fluid (BALF) of hamsters in the BL + DC group. However, DC treatment had little effect on BL-induced increases in lung superoxide dismutase activity and lipid peroxidation and leakage of plasma proteins in the BALF of the BL + DC group. Hamsters in the BL + PBS group showed severe multifocal fibrosis and accumulation of mononuclear inflammatory cells and granulocytes. In contrast, hamsters in the BL + DC group showed mild multifocal septal thickening with aggregations of mononuclear inflammatory cells. Hamsters in both control groups (SA + PBS and SA + DC) showed normal lung structure. Frozen lung sections following immunohistochemical staining revealed an intense staining for EDA-fibronectin and collagen type I in the BL + PBS group as compared with all other groups. It was concluded that DC potentially offers a novel pharmacological intervention that may be useful in treating pulmonary fibrosis.

Effect of antibody against integrin alpha4 on bleomycin-induced pulmonary fibrosis in mice.

Integrins are a family of transmembrane glycoproteins that can interact with components of the extracellular matrix. The alpha4beta1 and alpha4beta7 integrins are heterodimeric leukocyte cell surface molecules critical to their cell and matrix adhesive interactions. Evidence for a central role for the alpha4 integrins in leukocyte pathophysiology in the lung is well documented. In this study, we tested the hypothesis that neutralizing antibody for integrin alpha4 (PS2) may reduce bleomycin (BL)-induced lung fibrosis in vivo. Male C57BL/6 mice were injected intratracheally with saline (SA) or BL (0.08 U/mouse) followed by intraperitoneal injection of SA, isotype control antibody (1E6), or PS2 (100 microg) three times a week. Twenty-one days after the intratracheal instillation, mice were killed for bronchoalveolar lavage (BAL), biochemical, histopathological, and immunohistological analyses. Treatment with PS2 significantly reduced BL-induced increases in lung lipid peroxidation and hydroxyproline content. Lung histopathology also showed reduced fibrotic lesions in the BL-treated lungs by treatment with PS2. BL-treated mouse lungs also showed induction of cells with the myofibroblast phenotype, as indicated by the increased expression of alpha-smooth muscle actin (alphaSMA), whereas treatment with PS2 minimized the BL-induced alphaSMA expression. Furthermore, treatment with PS2 reduced the BL-induced increase in the BAL total cell number, and attenuated the BL-induced increase in the BAL protein level. It is concluded that integrin alpha4 may play an important role in BL-induced pulmonary fibrosis, and the use of anti-alpha4 antibody offers therapeutic antifibrotic potential in vivo.

Cellular and chemical mediators of type 1 hypersensitivity in calves infected with Ostertagia ostertagi: mast cells and eosinophils.

Abomasal mucosal mast cell and eosinophil accumulation was morphometrically evaluated in 26 Holstein steers after natural or experimental infection with Ostertagia ostertagi. Results showed that following infection, accumulation of mast cells and eosinophils in abomasal tissue was dependent on infection pattern. Eosinophilia was greater in steers with type 1 ostertagiosis, while mastocytes was more pronounced in steers with type 2 ostertagiosis.

Amelioration of bleomycin-induced lung fibrosis in hamsters by dietary supplementation with taurine and niacin: biochemical mechanisms.

Interstitial pulmonary fibrosis induced by intratracheal instillation of bleomycin (BL) involves an excess production of reactive oxygen species, unavailability of adequate levels of NAD and ATP to repair the injured pulmonary epithelium, and an overexuberant lung collagen reactivity followed by deposition of highly cross-linked mature collagen fibrils resistant to enzymatic degradation. In the present study, we have demonstrated that dietary supplementation with taurine and niacin offered almost complete protection against the lung fibrosis in a multidose BL hamster model. The mechanisms for the protective effect of taurine and niacin are multifaceted. These include the ability of taurine to scavenge HOCl and stabilize the biomembrane; niacin's ability to replenish the BL-induced depletion of NAD and ATP; and the combined effect of taurine and niacin to suppress all aspects of BL-induced increases in the lung collagen reactivity, a hallmark of interstitial pulmonary fibrosis. It was concluded from the data presented at this Conference that the combined treatment with taurine and niacin, which offers a multipronged approach, will have great therapeutic potential in the intervention of the development of chemically induced interstitial lung fibrosis in animals and humans.

Separation and spread of nuclear fragments ("nucleotesimals") in colonic neoplasms.

After multiple fragmentation of neoplastic nuclei into "nucleotesimals," a recently discovered variety of amitosis, some fragments appear to be inflated to full nuclear size; other probably lyse, releasing nucleic acid, which may assist in the synthesis of new cytoplasm around newly formed nuclei. This observation may be useful in elucidating one mechanism of neoplastic growth.

Quantitation of fibroblast population growth rate in situ using computerized image analysis.

The development of computer-assisted image analysis has provided the technology to rapidly determine the population size of cultured cell monolayers in situ. We have adapted this technology to determine the population growth rate of cultured fibroblasts for use in a high-replicate format. Human lung fibroblasts were seeded into 1/2 A 96-well plates that had one-half the culture area of standard 96-well plates. The cells were cultured in medium supplemented with different concentrations of FBS and on days 0, 1, 2, 3, 5, and 7, and their nuclei were stained with propidium iodide. A microscopic field representing one-quarter of a well of fluorescent nuclear images was captured onto a Macintosh computer, and the number of nuclei were counted using an image analysis software program. There were no significant differences between the number of nuclei counted manually and the number counted using computer-assisted software, until day 7 where the cells were multilayered (P < 0.05). This image analysis method was compared to other assays typically used to estimate cell proliferation or population size, namely hemocytometer counting, a rapid colorimetric staining assay using naphthol blue-black, and [3H]-thymidine incorporation. The growth rates derived using image analysis were in close agreement with results derived from hemocytometer counts and [3H]-thymidine incorporation. However, the growth rates of cells grown in high concentrations of FBS as determined using naphthol blue-black were substantially lower than results from image analysis. We conclude that this adaptation of computer-assisted image analysis provides a method to derive accurate growth curves by directly counting the number of cells in a large number of replicates.

Computer-assisted morphometry: point, intersection, and profile counting and three-dimensional reconstruction.

The use of computers in morphometry can involve 1) automated image analysis, semiautomated image analysis and point, intersection, intercept and profile counts of two-dimensional images on tissue sections with mathematical extrapolation to the third dimension, 2) direct measurement of volumes, surfaces, lengths, and curvature using x,y,z coordinates of serial sectioned images, or 3) stereologic techniques and serial sections which is a combination of 1 and 2 above. Automated and semiautomated image analysis are generally restricted to specimens that are characterized by differential contrast such as interalveolar septa in the lung or histochemically stained mucous granules in pulmonary epithelium. Point, intersection, and profile counts using hand-held, notebook PCs, portable PCs, or standard PCs and MS-DOS-based application programs are extremely efficient, precise, affordable, and convenient methods of quantitating average values of a population. When morphometric measurements of individual structures are required, computer-assisted three-dimensional reconstruction using x,y,z coordinates of the surface outline from serial sections is a tedious yet precise method. We describe a computer program that efficiently estimates mean caliper diameter, volume, and surface area with less than five percent error with five sections per structure. We also describe a program that does digital image subtraction on serial sections, superimposes digitally generated test systems on biological images, and accumulates point, intersection, and profile counts using a Macintosh II series computer.

Effect of C-fiber-mediated, ozone-induced rapid shallow breathing on airway epithelial injury in rats.

We examined the relationship between C-fiber-mediated, ozone-induced rapid shallow breathing and airway epithelial cell injury at different airway sites within the lower respiratory tract of conscious Wistar rats (n = 24). We combined an acute 8-h ozone inhalation with vagal perineural capsaicin treatment, a selective C-fiber conduction block, and 5-bromo-2'-deoxyuridine (BrdU) labeling as an index of epithelial injury. Vehicle-treated rats that inhaled ozone developed a rapid shallow breathing pattern during ozone inhalation, whereas the capsaicin-treated rats that inhaled ozone showed no changes in respiratory frequency. In vehicle-treated, ozone-exposed rats that developed rapid shallow breathing, a progressive increase in BrdU-labeling density (no. of BrdU-labeled cells/mm(2) airway) was observed starting at the bifurcation of the left main stem bronchi (central airway) and going down either a short or long airway path. In vehicle-treated, ozone-exposed rats, terminal bronchioles supplied by short and long airway paths had a similar degree of BrdU-labeling density that was significantly (P < 0.05) greater than the BrdU-labeling density of the proximal airways that supply them. In contrast, the attenuation of rapid shallow breathing produced by capsaicin treatment resulted in a significantly reduced BrdU-labeling density in the terminal bronchioles supplied by short airway paths compared with the terminal bronchioles supplied by long airway paths. Our data indicate that ozone-induced rapid shallow breathing protects large conducting airways while producing a more even distribution of injury to terminal bronchioles.

Capsaicin-sensitive C-fiber-mediated protective responses in ozone inhalation in rats.

To assess the role of lung sensory C fibers during and after inhalation of 1 part/million ozone for 8 h, we compared breathing pattern responses and epithelial injury-inflammation-repair in rats depleted of C fibers by systemic administration of capsaicin as neonates and in vehicle-treated control animals. Capsaicin-treated rats did not develop ozone-induced rapid, shallow breathing. Capsaicin-treated rats showed more severe necrosis in the nasal cavity and greater inflammation throughout the respiratory tract than did control rats exposed to ozone. Incorporation of 5-bromo-2'-deoxyuridine (a marker of DNA synthesis associated with proliferation) into terminal bronchiolar epithelial cells was not significantly affected by capsaicin treatment in rats exposed to ozone. However, when normalized to the degree of epithelial necrosis present in each rat studied, there was less 5-bromo-2'-deoxyuridine labeling in the terminal bronchioles of capsaicin-treated rats. These observations suggest that the ozone-induced release of neuropeptides does not measurably contribute to airway inflammation but may play a role in modulating basal and reparative airway epithelial cell proliferation.

Severe pulmonary hypertension and arterial adventitial changes in newborn calves at 4,300 m.

Some human newborns have a syndrome characterized by irreversible pulmonary hypertension and severe hypoxemia and by medial hypertrophy and adventitial thickening of pulmonary arteries. We considered that newborn calves made severely hypoxic might reproduce features of the human disease. When 2-day-old calves were placed at 4,300 m simulated altitude, pulmonary arterial pressure was increased and could be reversed by 100% O2. However, after 2 wk at 4,300 m, pulmonary arterial pressures were suprasystemic and there was right-to-left shunting probably through the foramen ovale and a patent but restrictive ductus arteriosus. Suprasystemic pulmonary pressure and hypoxemia persisted with 100% O2 breathing. Morphometrical examination of the lung arteries showed a markedly thickened adventitia with cellular proliferation and collagen and elastin deposition. There was increased medial thickness and distal muscularization of the pulmonary arteries associated with decreased luminal diameter. The rapid development of severe pulmonary hypertension and poor responsiveness to O2 was associated with increased arterial wall thickness, particularly involving the adventitia. Thus the pulmonary arterial circulation in these calves, which were placed at high altitude for 2 wk, exhibited features resembling persistent pulmonary hypertension in newborn infants.

Characterization of a phospholipase A2 in hamster lung and in vitro and in vivo effects of bleomycin on this enzyme.

Phospholipase A2 (PLA2) activity from adult hamster lung was characterized using L-alpha-1-palmitoyl-2-arachidonyl-[arachidonyl-1-14C]-phosphatidylcholine as the substrate. The released [14C]-arachidonic acid was separated by TLC. The enzyme activity increased with increasing incubation time (0-120 minutes), calcium ion concentration (0-25.0 mM) and protein (0-2.0 mg). The optimum pH was 8.0. Deoxycholate had a concentration dependent (0.1 to 0.5 mM) inhibitory effect on the activity. PLA2 specific activity was the highest in mitochondrial fraction. PLA2 activity following incubation with bleomycin was increased in a dose related fashion. In vivo study showed that both PLA2 activity and collagen content in hamster lung were significantly elevated at 14 days followed intratracheal instillation of bleomycin. The activation of PLA2 may play an important role in bleomycin-induced pulmonary toxicity.