Nitrofen Exposure Research Articles

Experimental congenital diaphragmatic hernia features an alteration of DNA sensing targets cGAS and STING.

The pathogenesis of congenital diaphragmatic hernia (CDH) depends on multiple factors. Activation of the DNA-sensing cyclic-GMP-AMP-synthase (cGAS) and Stimulator-of-Interferon-Genes (STING) pathway by double-stranded DNA (dsDNA) links environmental stimuli and inflammation. We hypothesized that nitrofen exposure alters cGAS and STING in human bronchial epithelial cells and fetal rat lungs. We used the Quant-IT™-PicoGreen™ assay to assess dsDNA concentration in BEAS-2B cells after 24 h of nitrofen-exposure and performed immunofluorescence of cGAS/STING. We used nitrofen to induce CDH and harvested control and CDH lungs at embryonic day E15, E18 and E21 for cGAS/STING immunofluorescence, RT-qPCR and RNA-Scope™ in-situ-hybridization (E18, E21). We found a higher concentration of dsDNA following nitrofen treatment. Nitrofen-exposure to BEAS-2B cells increased cGAS and STING protein abundance. cGAS abundance was higher in nitrofen lungs at E15, E18 and E21. RNA-Scope in-situ-hybridization showed higher cGAS and STING expression in E18 and E21 lungs. RT-qPCR revealed higher mRNA expression levels of STING in E21 nitrofen-induced lungs. Our data suggest that nitrofen-exposure increases dsDNA content which leads to stimulation of the cGAS/STING pathway in human BEAS-2B cells and the nitrofen rat model of CDH. Consequently, DNA sensing and the cGAS-STING-pathway potentially contribute to abnormal lung development in CDH. We found an alteration of DNA sensing targets cGAS and STING in human BEAS-2B cells and experimental congenital diaphragmatic hernia with higher protein abundance and mRNA expression in cells and lung sections of nitrofen-treated rat pups. This is the first study to investigate DNA sensing, a potential link between environmental stimuli and inflammation, in experimental CDH. Our study extends the knowledge on the pathogenesis of experimental CDH.

Proteomic Profiling of Hypoplastic Lungs Suggests an Underlying Inflammatory Response in the Pathogenesis of Abnormal Lung Development in Congenital Diaphragmatic Hernia.

The pathogenesis of lung hypoplasia in congenital diaphragmatic hernia (CDH), a common birth defect, is poorly understood. The diaphragmatic defect can be repaired surgically, but the abnormal lung development contributes to a high mortality in these patients. To understand the underlying pathobiology, we compared the proteomic profiles of fetal rat lungs at the alveolar stage (E21) that were either exposed to nitrofen in utero (CDH lungs, n=5) or exposed to vehicle only (non-CDH control lungs, n=5). Pathway analysis of proteomic datasets showed significant enrichment in inflammatory response proteins associated with cytokine signaling and Epstein Barr Virus in nitrofen CDH lungs. Among the 218 significantly altered proteins between CDH and non-CDH control lungs were Tenascin C, CREBBP, LYN, and STAT3. We showed that Tenascin C was decreased around the distal airway branches in nitrofen rat lungs and human CDH lungs, obtained from stillborn fetuses that did not receive pre- or postnatal treatment. In contrast, STAT3 was significantly increased in the airway epithelium of nitrofen lungs at E21. STAT3 inhibition after direct nitrofen exposure to fetal rat lung explants (E14.5) partially rescued the hypoplastic lung phenotype ex vivo by increasing peripheral lung budding. Moreover, we demonstrated that several STAT3-associated cytokines (IL-15, IL-9, andIL-2) are increased in fetal tracheal aspirates of CDH survivors compared with nonsurvivors after fetoscopic endoluminal tracheal occlusion. With our unbiased proteomics approach, we showed for the first time that downstream inflammatory processes are likely involved in the pathogenesis of abnormal lung development in CDH.

Assessment of the nitrofen model of congenital diaphragmatic hernia and of the dysregulated factors involved in pulmonary hypoplasia.

To study pulmonary hypoplasia (PH) associated with congenital diaphragmatic hernia (CDH), investigators have been employing a fetal rat model based on nitrofen administration to dams. Herein, we aimed to: (1) investigate the validity of the model, and (2) synthesize the main biological pathways implicated in the development of PH associated with CDH. Using a defined strategy, we conducted a systematic review of the literature searching for studies reporting the incidence of CDH or factors involved in PH development. We also searched for PH factor interactions, relevance to lung development and to human PH. Of 335 full-text articles, 116 reported the incidence of CDH after nitrofen exposure or dysregulated factors in the lungs of nitrofen-exposed rat fetuses. CDH incidence: 54% (27-85%) fetuses developed a diaphragmatic defect, whereas the whole litter had PH in varying degrees. Downregulated signaling pathways included FGF/FGFR, BMP/BMPR, Sonic Hedgehog and retinoid acid signaling pathway, resulting in a delay in early epithelial differentiation, immature distal epithelium and dysfunctional mesenchyme. The nitrofen model effectively reproduces PH as it disrupts pathways that are critical for lung branching morphogenesis and alveolar differentiation. The low CDH rate confirms that PH is an associated phenomenon rather than the result of mechanical compression alone.

Impaired VEGF and nitric oxide signaling after nitrofen exposure in rat fetal lung explants

We hypothesized that abnormal fetal lung growth in experimental congenital diaphragmatic hernia after maternal nitrofen exposure alters lung structure due to impaired VEGF signaling, which can be reversed with VEGF or nitric oxide (NO) treatment. Timed-pregnant Sprague-Dawley rats were treated with nitrofen on embryonic day 9 (E9), and fetal lungs were harvested for explant culture on E15. Explants were maintained in 3% O2 for 3 days and were treated with NO gas or recombinant human VEGF protein for 3 days. To determine the effects of VEGF inhibition on lung structure, normal fetal lung explants were treated with SU-5416, a VEGF receptor inhibitor, with or without exogenous NO or VEGF. We found that nitrofen treatment impaired lung structure, as evidenced by decreased branching at day 0, but lung structure was not different from controls after 3 days in culture. Nitrofen reduced lung VEGF but not endothelial NO synthase protein level. Treatment with NO enhanced lung growth in control and nitrofen-exposed lungs; however, the response to NO in the nitrofen-treated lungs was reduced when compared with controls. VEGF treatment did not cause a further increase in lung complexity after nitrofen exposure. SU-5416 treatment altered lung structure, which improved with NO but not VEGF treatment. Both nitrofen and SU-5416 treatment increased apoptosis in the mesenchyme of fetal lung explants. We conclude that nitrofen exposure increased apoptosis, decreased lung growth and reduced VEGF expression, and that exogenous NO but not VEGF treatment enhances lung growth. Disruption of lung architecture after VEGF receptor blockade was similar to nitrofen-induced changes but was more responsive to NO.

CFTR gene therapy, a method to rescue lung hypoplasia in congenital diaphragmatic hernia?

despite progress in neonatal care and despite changing concepts in treatment in the recent years, the mortality rate of infants with congenital diaphragmatic hernia (CDH) remains high ([20][1]). Pulmonary hypoplasia and vascular alterations associated with CDH have tremendous postnatal functional

Improvement of pulmonary hypoplasia associated with congenital diaphragmatic hernia by in utero CFTR gene therapy

Congenital diaphragmatic hernia (CDH) may be an ideal candidate disease for in utero gene therapy as disrupted fetal lung growth plays a significant role in disease outcome. We previously demonstrated that transient in utero overexpression of CFTR during fetal development resulted in lung epithelial proliferation and differentiation. We hypothesized that gene therapy with CFTR would improve the pulmonary hypoplasia associated with congenital diaphragmatic hernia (CDH). CDH was induced by the herbicide 2,4-dichlorophenyl-4-nitrophyl ether (nitrofen) following maternal ingestion at either 10 or 13 days gestation. In utero gene transfer of the CFTR gene was subsequently performed at 16 days gestation. Examination of the fetuses at 22 days gestation revealed little improvement in the CFTR-treated lungs following induction of hernias with nitrofen at 10 days gestation. However, the CFTR gene treatment significantly improved internal surface area, saccular density, overall saccular number, and amount of saccular air space in the lungs that were treated with nitrofen at 13 days gestation. RT-PCR demonstrated that gene transfer occurred following treatment at 13 days gestation but not in the lungs treated with nitrofen at 10 days gestation, despite gene transfer at the same gestational age (16 days) in both groups. As disruption of lung development correlates with the gestational stage at which nitrofen exposure occurs, these results confirmed previous findings that in utero gene transfer efficiency depends on the stage of lung development. Lung development may be significantly delayed in human CDH to allow for successful gene transfer later in gestation, providing a substantial therapeutic window.

Sex and congenital diaphragmatic hernia

Human studies note sex reversal syndromes and sex difference(s) in the incidence of congenital diaphragmatic hernia (CDH). Epidemiology surveys record a higher incidence of CDH in females, whilst other reports cite a higher frequency in males. Nitrofen, a teratogen, produces experimental CDH. This agent is speculated to interfere with retinoid acid-steroid signalling pathways and may also be linked with sexual differentiation. This study was designed therefore to test the hypothesis that nitrofen may influence sexual phenotype and frequency of CDH. Time mated Sprague Dawley rats were dosed with nitrofen at day 9.5 to generate predominantly left sided CDH. Fetuses were delivered by caesarean section on days 20 or 21 of gestation (term=day 22). External genitalia were examined to define external genital phenotype. The abdominal cavity was opened and the genito-urinary system carefully examined. The internal genital organs were assigned a phenotype and findings correlated with external appearances. The diaphragm of each fetus was studied for the absence or presence of CDH and the laterality of defect recorded. Controls (non nitrofen fed) were used for all comparative analysis. Control (n=600) and nitrofen exposed offspring (n=504) had equal frequencies of males and females. CDH occurred with similar incidence in male and female nitrofen treated pups. In all nitrofen exposed fetuses and normal controls, internal and external genitalia concorded without evidence of significant genital tract malformations or intersex states. Prenatal nitrofen exposure is not associated with significant gender differences (or prenatal loss) in the risk of CDH. Genital tract malformations do not appear to accompany CDH in the nitrofen model.

The relationship of diaphragmatic defect, liver growth, and lung hypoplasia in nitrofen-induced congenital diaphragmatic hernia in the rat.

Reduced lung size (lung hypoplasia, LH) is the main cause of mortality in newborns with congenital diaphragmatic hernia (CDH). However, it is unclear which mechanisms lead to LH. To assess this, we analyzed the relationship of LH and liver mass in correlation to the size of the diaphragmatic defect in rats with nitrofen-induced CDH. A total of 266 newborn Sprague-Dawley rats (30 litters) were exposed to nitrofen on day 11.5 of pregnancy. After spontaneous delivery at term (22 days), all newborns were microdissected. Using a computerized morphometric device, the area of the thoracic cavity, the lung, the intrathoracic liver, and the diaphragmatic defect were measured. The lungs, the intrathoracic, and the extrathoracic portion of the liver were weighed. After nitrofen exposure, 160 newborn rats presented with CDH (60.2%). They were divided into five groups according to the intrathoracic content of intraabdominal organs. We observed a significant increase of the total liver and decrease of the lung weight in the severely affected groups. A significant correlation between the size of the defect and the weight of the intrathoracic part of the liver could be demonstrated. Nitrofen alone had no effect on liver weight. Our results indicate that (1) the presence of liver inside the thoracic cavity is not the result of dislocation but rather of growth of liver tissue through the defect, and (2) the observed correlation between the size of the defect and the intrathoracic liver weight may be part of the pathogenesis of LH in CDH.

Exposure of cardiac fibroblasts to the herbicide nitrofen causes altered interactions with the extracellular matrix.

A large proportion of congenital heart defects result from dysmorphogenesis of valvuloseptal precursors, the endocardial cushions. Intrinsic to formation and maturation of these tissues are developmental changes in cell-cell and cell-extracellular matrix interactions. Interactions between cells and the extracellular matrix play critical roles in modulating cellular processes including proliferation, migration, differentiation and even survival. While significant progress is being made in the elucidation of the cellular events involved in valvuloseptal development, little is known regarding how environmental factors may affect this process. Embryonic exposure to the herbicide nitrofen has been shown to result in congenital heart defects associated with altered endocardial cushion formation or maturation. The present studies were performed to begin to address the cellular mechanisms of these nitrofen-induced effects. Heart fibroblasts were isolated and treated with varying doses of nitrofen in vitro. Experiments were performed to determine the effects of this herbicide on important cellular processes including migration, proliferation and apoptosis. These studies illustrated a dose-dependent decrease in collagen gel contraction and proliferation in response to nitrofen. Assays were also performed to determine the effects of nitrofen on fibroblast gene expression. Increased expression of collagen type I and specific integrins were seen following nitrofen exposure. These studies illustrate that nitrofen has direct effects on cardiac fibroblast proliferation and extracellular matrix remodeling, cellular events important in valvuloseptal development.

Murine nitrofen-induced pulmonary hypoplasia does not involve induction of TGF-β signaling

Background/Purpose: In the murine nitrofen-induced model of congenital diaphragmatic hernia (CDH), the lungs are primarily hypoplastic and immature even before diaphragmatic closure. Because excess transforming growth factor-β (TGF-β) signaling induces pulmonary hypoplasia, the authors hypothesized that primary hypoplasia after nitrofen exposure may be caused by abberant signaling by the TGF-β pathway. Therefore, abrogation of TGF-β signaling might rescue the hypoplasia. Methods: The authors performed intratracheal microinjections of a recombinant adenoviral vector encoding a dominant-negative TGF-β type II receptor (AdIIR-DN) in nitrofen-exposed and control E12 mouse lungs, which then were cultured for 4 days in serumless chemically defined media. The mRNA expression of Smad2, 3, 4, and 7 in nitrofen-exposed and control E12 lungs after 4 days in culture were compared. Results: ADIIR-DN increased terminal branching in control lungs by 28% compared with lungs injected with control virus (61.8 ± 4.6 v. 48.4 ± 4.7, P =.004). However, there was no difference between nitrofen-exposed lungs injected with ADIIR-DN and those injected with control virus. Compared with control lungs, Smad mRNA expression was decreased markedly in nitrofen-exposed lungs: Smad2 (40%, P =.16), Smad3 (29%, P =.02), Smad4 (25%, P =.07), and Smad7 (36%, P =.04). Conclusions: Because abrogation of TGF-β signaling does not rescue the hypoplasia seen in the nitrofen model, and Smad expression is decreased in nitrofen-exposed lungs, the TGF-β pathway does not appear to play a role in nitrofen-induced pulmonary hypoplasia. J Pediatr Surg 37:1123-1127. Copyright 2002, Elsevier Science (USA). All rights reserved.

Down-regulation of regulatory proteins for differentiation and proliferation in murine fetal hypoplastic lungs: altered mesenchymal-epithelial interactions.

We compared proliferation (growth) and differentiation (development) related proteins in normal and hypoplastic fetal murine lungs. The hypoplastic lungs were created in CD-1 fetal mice by nitrofen exposure (25 mg per pregnant mouse given intragastrically on gestational day 8 [Gd8]), as published earlier. The lungs were harvested at Gd14, 16, 19 and from neonates. Immunoblot analyses were carried out for transcription factors (oncogenic proteins, nuclear receptor, and transmembrane receptor proteins) in severely hypoplastic murine fetal lungs with coexistent diaphragmatic hernia, and results were compared with those derived from normal lungs of equivalent age. These proteins have proposed roles in the regulation of proliferation and differentiation processes of fetal lungs. We have shown that the product of the oncogene c-myc was reduced in hypoplastic lungs at all stages of gestation, whereas c-Fos protein levels were variable. These proteins are known to regulate transcription of various developmental proteins, such as those responsible for proliferation and differentiation. Further, the nuclear transcription factors thyroid transcription factor-1 (TITF-1) and glucocorticoid receptor (GR) were reduced, and thyroid hormone receptor (TR) and retinoic acid receptors (RARs) were inhibited in severely hypoplastic lungs compared to normal lungs of equivalent gestational stage, except in neonatal lungs, where signals for RARs were seen. TITF-1 is known to localize in bronchial epithelial cells in developing lungs. It is restricted to type II pneumocytes with gestational development in the normal lungs and regulates surfactant proteins. Earlier, we have reported that surfactant proteins are reduced in hypoplastic lungs. In the current study, reduced GR and TITF-1 proteins may play a role in reducing surfactant proteins in the hypoplastic lungs. The significant inhibition in TR and RARalpha in the severely hypoplastic lungs reflects on affected epithelial cell maturation and alveolar formation, respectively. Altered RARbeta levels correlate with affected lung growth and branching morphogenesis of nitrofen-exposed lungs. A transmembrane receptor protein EGFR was reduced in hypoplastic lungs, suggesting the involvement of altered mesenchymal-epithelial signal transduction pathways. We conclude (1) Our data suggest altered levels of various nuclear transcription factors in the murine fetal hypoplastic lungs; (2) Reduced levels TITF-1 protein in hypoplastic lungs may have caused the functional immaturity of distal lung, immature airways and thus may affect overall differentiation of lungs. These results correlated with low levels of surfactant proteins in these lungs; (3) TR and RAR inhibition indicate their roles through reduced or retarded proliferation and differentiation processes in the severely hypoplastic lungs; (4) GR down-regulation in developing fetal murine hypoplastic lungs indicate delayed development, and GR up-regulation in affected neonates may be induced by stress/stretch caused at birth due to air-breathing; (5) Down- regulation of EGFR indicate altered mesenchymal-epithelial interactions and possible influence on lung proliferation and differentiation.

Prenatal exposure to nitrofen induces Fryns phenotype in mice.

Prenatal exposure to nitrofen is known to cause multiple malformations in mice. The reported malformations include lung hypoplasia, diaphragmatic hernia, cardiovascular defects, skeletal malformations, cleft palate, and renal abnormalities. The authors present detailed findings of craniofacial defects after prenatal exposure to nitrofen, and propose that together with the previously reported malformations, nitrofen exposure induces a Fryns phenotype in mice. Fryns syndrome is a rare human genetic syndrome that is an autosomal recessive disorder characterized by lung hypoplasia, diaphragmatic hernia, craniofacial malformations, skeletal malformations, cardiovascular malformations, and genitourinary malformations. Timed-pregnant Swiss Webster mice were gavage-fed 25 mg of nitrofen on day 8 of gestation. Control animals received olive oil. Osteogenesis and chondrogenesis were studied in fetuses recovered on day 17 after Alcian blue-Alizarin red staining. Approximately 26% of the nitrofen-exposed embryos had severe craniofacial defects, and there was generalized delay in chondrogenesis and osteogenesis throughout the skeleton. No such defects were noted in the control group. The authors propose that prenatal exposure to nitrofen induces a Fryns phenotype in mice, and thus speculate that nitrofen may target similar molecular mechanisms to those that lead to Fryns syndrome.

Surfactant protein A is decreased in a rat model of congenital diaphragmatic hernia.

We hypothesized that the expression of surfactant protein A (SP-A) would be altered in developing lungs from rat fetuses with congenital diaphragmatic hernia (CDH) induced by maternal ingestion of 2,4-dichlorophenyl-p-nitrophenyl ether (Nitrofen) on Day 9 of gestation. We compared our findings in fetuses exposed to Nitrofen with a CDH with those in Nitrofen-exposed fetuses without a CDH, and control fetuses whose mothers received olive oil only, the vehicle for Nitrofen. In late gestation, immunocytochemistry using a polyclonal rabbit antihuman SP-A antibody revealed decreased amounts of this protein in lungs from fetuses with CDH. Using immunoblotting, the relative amount of SP-A on Day 21 of gestation was also decreased in lung tissue from fetuses with CDH compared with the other groups. Abnormalities of mRNA for SP-A were observed in both groups of Nitrofen-exposed fetuses compared with control rats. These findings suggest that there is decreased expression of SP-A in rat fetuses with CDH secondary to Nitrofen exposure.

Intestinal rotation in experimental congenital diaphragmatic hernia

This study examines whether experimental congenital diaphragmatic hernia (CDH) induced by nitrofen in rats is accompanied by intestinal malrotation similar to that observed in the human condition. Time-dated pregnant rats were fed 100 mg of nitrofen on day 9.5 of gestation, and fetuses were examined on days 17, 19, and 21. Body weight, lung weight, grade of bowel herniation into the umbilical cord and grade of intestinal malrotation were compared with those of age-matched controls. Body and lung weights were significantly lower in nitrofen-exposed rats on days 17, 19, and 21. The umbilical hernia persisted on day 17 in 100% of experimental animals and 66% of controls ( P<.01). Intestinal malrotation was more severe in experimental rats than in controls on days 19 (63% v 17% grade 2; P<.01) and 21 (27% v 0% grade 1; P<.01). Finally, 52% of nitrofen-fed fetuses with CDH had malrotation at term, whereas only 18.2% of those without it did ( P<.05). There was a significant ( P<.001) negative correlation between the lung weight/body weight ratio and the degree of malrotation in nitrofentreated fetuses. In conclusion, maternal nitrofen exposure on gestational day 9.5 induces intestinal malrotation in fetal rats by (1) delaying fetal growth and maturation; (2) causing CDH, which permits displacement of the liver and the gut into the thorax during the critical period of reintegration and fixation; and (3) inducing lung hypoplasia and reducing thoracic volume during this period.

265 EARLY LUNG DEVELOPMENT IN THE RAT AND ITS PERTURBATION BY NITROFEN EXPOSURE

In search for mechanisms underlying pulmonary hypoplasia we have studied early lung development in the rat and its perturbation by Nitrofen exposure using serially sectioned embryos. The lung primordium is visible from day 12 of gestation, firstly as an outgrowth of the bottom of the larynx, and on day 13 as a triplicate tube growing in caudal direction. The longitudinal growth speed of the lungs exceed the growth of the whole embryo, resulting on day 15 in a longitudinal lung length of 20% of the embryo. During this period the lung primordium, is built up of undifferentiated epithelium surrounded by mesenchymal tissue. Exposure to Nitrofen on day 10 reduces the early proliferation and growth of the lung primordium, as measured in serial longitudinal sections of treated embryos. In fetuses that did not develop diaphragmatic hernia (DH) lung lenth had recovered to control range by day 20, irrespective of the Nitrofen dosage applied. In fetuses with leftsided DH, leftsided lung hypoplasia occurred in conjunction with right lungs that were significantly longer than controls. The results suggest two determinants of development of lung hypoplasia in this model; an early effect on proliferation of the lung primordum, and a late effect related to the presence of DH.

Alveolar epithelial composition and architecture of the late fetal pulmonary acinus: an immunocytochemical and morphometric study in a rat model of pulmonary hypoplasia and congenital diaphragmatic hernia.

The aim of the present study was to compare the architecture and alveolar epithelial cell composition of the pulmonary acinus in hypoplastic and normal fetal rat lungs. For this purpose, a rat model of pulmonary hypoplasia in association with congenital diaphragmatic hernia (CDH) induced by Nitrofen (100 mg on day 10 of pregnancy) was studied. Sections (5 microns) from lungs of control and Nitrofen-exposed fetal Sprague Dawley rats with or without CDH aged 18-22 days (vaginal plug on day 1, birth on day 23) were stained with hematoxylin and eosin. To identify developing alveolar epithelial cells, sections were incubated with anti-surfactant protein A (SP-A; rabbit anti-mouse) or preimmunization serum (indirect immunofluorescence). On days 18 and 19, control lungs and exposed lungs from fetuses with and without CDH looked similar (pseudoglandular stage of lung development). The prospective pulmonary acinus consisted of acinar tubules with small round lumens, lined by cuboid, fluorescent type II cells. Morphometric analysis on day 19 showed significantly smaller lung volumes and lung tissue volumes after Nitrofen exposure. On day 20 (canalicular stage), some tubules were slightly dilated and lined by cuboid and thinner fluorescent cells; these dilated tubules were less numerous in lungs from exposed fetuses with CDH. On days 21 and 22 (saccular stage), the saccular lining consisted of cuboid to thin fluorescent cells in exposed lungs from fetuses with and without CDH, and fluorescent (low) cuboid cells interspersed with dark zones (type I cell areas) in control lungs. In the exposed lungs from fetuses with CDH, the lumens of all airspaces were frequently slit-like, and the septa were thicker. These phenomena gave the lungs a primitive, compact aspect. Morphometric analysis on day 22 showed smaller lung volumes and lung tissue volumes, smaller airspace/tissue ratios, smaller epithelial surface areas, and more type II cells per surface area in Nitrofen-exposed lungs than in normal control lungs. The results suggest that Nitrofen-exposed, and thus hypoplastic, fetal rat lungs are retarded with respect to the differentiation of cuboid type II cells into squamous type I cells whether or not CDH is present, and with respect to the development of the future airspaces between days 20 and 22 if CDH is present.

Developmental toxicity of diphenyl ether herbicides in nestling American kestrels

Beginning the day after hatching, American kestrel (Falco sparverius) nestlings were orally dosed for 10 consecutive days with 5 microliters/g of corn oil (controls) or one of the diphenyl ether herbicides (nitrofen, bifenox, or oxyfluorfen) at concentrations of 10, 50, 250, or 500 mg/kg in corn oil. At 500 mg/kg, nitrofen resulted in complete nestling mortality, bifenox in high (66%) mortality, and oxyfluorfen in no mortality. Nitrofen at 250 mg/kg reduced nestling growth as reflected by decreased body weight, crown-rump length, and bone lengths including humerus, radius-ulna, femur, and tibiotarsus. Bifenox at 250 mg/kg had less effect on growth than nitrofen, but crown-rump, humerus, radius-ulna, and femur were significantly shorter than controls. Liver weight as a percent of body weight increased with 50 and 250 mg/kg nitrofen. Other manifestations of impending hepatotoxicity following nitrofen ingestion included increased hepatic GSH peroxidase activity in all nitrofen-treated groups, and increased plasma enzyme activities for ALT, AST, and LDH-L in the 250-mg/kg group. Bifenox ingestion resulted in increased hepatic GSH peroxidase activity in the 50- and 250-mg/kg groups. Nitrofen exposure also resulted in an increase in total plasma thyroxine (T4) concentration. These findings suggest that altricial nestlings are more sensitive to diphenyl ether herbicides than young or adult birds of precocial species.

Effects of prenatal nitrofen exposure on cardiac structure and function in the rat

The herbicide nitrofen was administered to pregnant Fischer-344 and Sprague-Dawley rats on Days 10–13 of gestation (po, 20 or 40 mg/kg daily) and its effects on cardiac structure and function were investigated in the offspring. In the 21-day fetuses, nitrofen did not influence intrauterine growth or basal heart rate. In contrast, the herbicide produced a marked depression of heart rate and abnormal electrocardiographic (ECG) profiles in the newborn rats, in conjunction with labored respiratory movements and a profound increase in postnatal mortality. A few animals displayed cardiac ventricular septal defects and diaphragmatic hernias but these malformations did not appear to be associated with the ECG changes. The chronotropic deficiencies seen in the nitrofen-treated pups were reversible by acute hyperoxia (40% oxygen). These results suggest that the teratogenic effects of nitrofen on cardiac physiology and on postnatal mortality cannot be accounted for solely by specific gross anatomical damages to the rat heart and diaphragm; rather, other more subtle morphological and physiological factors which contribute to improper systemic delivery and cellular utilization of oxygen may be involved.

Kidney morphology and function in the young of rats malnourished and exposed to nitrofen during pregnancy.

The separate and combined effects of prenatal protein deficiency (6% casein) and prenatal nitrofen (2,4-dichlorophenyl-p-nitrophenyl ether) exposure (12.5 mg/kg on gestational d 7-21) on renal morphology in the 21-d fetal and postnatal rat were examined. Body weights and kidney weights were reduced in prenatally protein-deprived (PPD) pups at birth and on postnatal day (PND) 10. Numbers of mature glomeruli, creatinine clearance, water diuresis, and response of antidiuretic hormone (ADH), but not the concentrating ability, were lower in the PPD neonates. These changes suggest that prenatal protein deficiency delays renal development and possibly results in a decrease in glomerular clearance and in tubular response to a water load and to antidiuretic hormone. Prenatal nitrofen exposure reduced body weight and kidney size on PND 0 and 10. An increased incidence of hydronephrosis was indicated in the nitrofen-exposed fetus. Prenatal nitrofen exposure depressed the ability to excrete excess water, the response to ADH, and urine-concentrating ability. The functional deficits indicate tubular dysfunction, but little or no effect on glomerular function, as indicated by the absence of an effect on creatinine clearance. Postnatal survival was reduced to 22% by PND in the PPD plus nitrofen pups. Also, prenatal nitrofen exposure increased the susceptibility of the glomeruli in the gestational day (GD) 21 PPD fetus to the adverse effects of prenatal protein deficiency. By PND 10 the toxic effects were of the same order. Renal dysfunction may contribute to the increased mortality in PPD plus nitrofen pups by reducing the ability to respond to stress, but the effects are not sufficiently marked to be considered the primary cause of death.

Effects on the fetus of maternal nitrofen exposure in the protein-deprived rat

The separate and combined effects of protein deprivation and nitrofen exposure were studied in the pregnant rat. Animals were fed diets containing 24, 8, 6 or 4% casein throughout gestation. Within each diet group, subgroups were gavage-fed with 12.5 (lower dose and 25 (higher dose) mg nitrofen/kg body weight or with oil carrier only on days 7–21 of gestation. Dams were weighed and food intake was measured daily. On day 21 of gestation, cesarean-derived pups were examined for congenital anomalies and dissected for determination of organ weights. Skeletons were alizarinstained and examined for skeletal anomalies and developmental stage. No effects of the skeleton or gross congenital anomalies were seen. Fetal size and weights of liver, kidney, intestine, heart, lung and brain were reduced with decreasing casein content of the diet and as a result of the higher dose of nitrofen. An effect of interaction between diet and nitrofen exposure was shown in the kidney, intestine and lung weights. Specific toxicity affecting organ size was shown to occur in the intestine and lung. An interaction between diet and nitrofen specifically affected kidney and intestine. Brain size tended to be preserved, a possible protective mechanism negated by nitrofen exposure. The data suggest also that kidney, intestine and lung are particularly affected. Effects, however, occur primarily in young of severely malnourished dams receiving a relatively high dose of nitrofen.