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Intraperitoneal injection of 5 mg or 10 mg per kg body weight of technical grade lindane twice weekly in Charles-Foster female rats induced marked disturbance of the estrous cycle, prolonging the proestrus phase considerably (5-7 times more compared to the normal) and thereby delaying ovulation. Exfoliated cells at proestrus are marked by high concentrations of glycogen, SDH and alkaline phosphatase while estrus is characterized by increased activity of acid phosphatase and mucopolysaccharides. Corresponding changes in the polysaccharides concentrations and enzyme activities of the exfoliated cells seen in the lindane treated rats were well correlated with disturbances of the cycle. The data suggest that lindane exerts an estrogen-like effect by inducing a temporary negative feedback on pituitary gonadotropin secretion.

Residue Accumulation in Mice Chronically Fed Lindane (?, HCH)

Objective To investigate the lung response to traffic-related air pollution by enumerating hemosiderin-laden alveolar macrophages (AM) in sputum. Study design Sputum samples were collected from 103 urban adult males from Calcutta chronically exposed to automobile exhaust. Forty-nine rural individuals served as controls. AM were identified by nonspecific esterase staining. Perl's Prussian blue technique was employed for the detection of hemosiderin-laden AM (siderophages). Results The urban group, consisting of 31 traffic officers, 25 automobile service station workers and 47 street hawkers, had seven times more AM in their sputum than did the matched controls. Besides, a remarkable rise (27-fold) in the number of siderophages in sputum was observed in urban individuals. Smoking further elevated the AM count and number of siderophages. Conclusion Abundant siderophages in the urban group may indicate the toxic effect of airborne pollutants on the lung, leading to phagocytosis of destroyed cells, including erythrocytes, and accumulation of iron in AM. Enumeration of siderophages in sputum appears to be a simple, noninvasive, inexpensive cytochemical technique well suited to preliminary assessment of the adverse effects of air pollution on the lungs in large, population-based studies, especially in developing countries.

1. No difference was observed in thein vitro growing ability of granule cells isolated from hypothyroid or normal rat brain. When granule cells were taken from hypothyroid rat brain and grown in normal culture medium containing 10% fetal calf serum, they behaved similarly to the granule cells obtained from normal rat brain. 2. In both cases there were progressive losses ofin vitro growing ability of the granule cells with the age of the animal and it became impossible to grow them when derived from 21 days or older animals. 3. A marked decrease in cell surface GM1 was observed when the cells were maintained under thyroid hormone-deficient conditions in culture. 4. Anti-GM1 antibody was found to inhibit significantly the migration of granule cells along the astrocyte fibers. 5. These results indicate that GM1 has an important role in thyroid hormone-dependent postnatal brain maturation in rat.

Hepatic bilirubin excretion requires UDP-glucuronosyltransferase-mediated glucuronidation. Patients with type I Crigler-Najjar syndrome and mutant rats (Gunn strain) inherit deficiency of UDP-glucuronyltransferase activity toward bilirubin as an autosomal recessive trait and, as a result, exhibit marked nonhemolytic unconjugated hyperbilirubinemia throughout postnatal life. Heterozygous carriers of the trait have normal serum bilirubin levels. Because of placental excretion of unconjugated bilirubin, type 1 Crigler-Najjar syndrome patients and Gunn rats are not jaundiced in utero, making prenatal diagnosis difficult. Here we report a diagnostic method in Gunn rats based on genomic DNA analysis for prenatal recognition of deficiency of UDP-glucuronyltransferase activity toward bilirubin in Gunn rats and identification of heterozygous carriers. We and others have shown that two distinct messenger RNA species (UDP-glucuronyltransferase activity toward bilirubin and the 3-methylcholanthrene-inducible phenol-UDP-glucuronyltransferase messenger RNA) in Gunn rat liver contain identical deletions of a single guanosine residue in their common 3' regions. Loss of the restriction site for the endonuclease BstNI, which results from this deletion, was used as the basis for a diagnostic test. Female heterozygous Gunn rats were mated with male homozygous Gunn rats. Genomic DNA was extracted from the chorionic aspect of placenta of 17-day fetuses or from leukocytes from normal rats, obligate heterozygotes and homozygous Gunn rats. The DNA was sequentially digested with the restriction enzymes EcoRI and BstNI and subjected to Southern-blot analysis with a double-stranded DNA probe for the common region of UDP-glucuronyltransferase activity toward bilirubin and the 3-methylcholanthrene-inducible UDP-glucuronyltransferase messenger RNAs.(ABSTRACT TRUNCATED AT 250 WORDS)

Rat brain, obtained 10 min after death, contained high levels of endogenous gamma-aminobutyric acid (GABA) and glutamic acid. Incubation of this brain homogenate at 37 degrees C indicated decrease of GABA with time due to degradation by GABA-transaminase. Reported high-performance liquid chromatographic (HPLC) methods for glutamic acid decarboxylase (GAD) assay depend on the difference between the GABA content of the reaction mixture after and before the incubation period. None of the methods considered the degradation of GABA during incubation. Furthermore, during determination of the Michaelis constant (KM) for the reaction none of them considered the endogenous substrate. Here we have focused on these factors which seriously affect the maximum velocity (Vmax) and KM values during GAD assay by the HPLC technique. By a simple and rapid HPLC technique we have measured GAD activity in post-mortem rat brain after removing endogenous glutamic acid by charcoal treatment and using gabaquline to prevent GABA degradation during incubation period. By this method a Vmax value of 46 +/- 4 nmol/h/mg protein and a KM value of 7.5 +/- 0.6 mM were observed for GAD activity of crude brain homogenate. For a comparative study, we have carried out radiometric assay of GAD activity from the same sample and observed a Vmax of 48 +/- 6 nmol/h/mg protein and KM of 6.9 +/- 0.4 mM.

Lindane (γ-HCH) given to adult female mice orally at various doses and over varying periods adversely affected adrenocortical function. Adrenal weights decreased and both fasciculata and reticularis zones markedly regressed. Histopathological lesions were also noted and plasma and glandular glucocorticoid contents were significantly reduced. Simultaneously an increase in cholesterol and a decrease in ascorbic acid content of the adrenal glands were noticed. The adrenotoxic effect of this chlorinated insecticide possibly results from depressed corticoid production in situ and /or suppressed activity of enzyme(s) that catalyze the peripheral transformation of steroids.

Crigler-Najjar syndrome, type I (CN-I) is a potentially lethal disorder characterized by severe unconjugated hyperbilirubinemia resulting from a recessively inherited deficiency of hepatic UDP-glucuronosyl-transferase (UGT) activity toward bilirubin (B-UGT). Two forms of B-UGT exist in human liver. mRNAs for these two forms and that for another isoform with activity toward simple phenols (P-UGT) have unique 5' regions, but their 3' regions are identical. The three mRNA species are derived from a single locus; the unique 5' regions are encoded by single unique exons and the identical 3' regions consist of four consecutive exons that are shared by all three isoforms. In this paper, we determined genetic lesions in two CN-I patients with deficiency of hepatic B-UGT and P-UGT activities. In one patient, there was a C----T substitution in exon 4 (common region) predicting the substitution of a serine residue with a phenylalanine residue; this mutation was present in the identical region of B-UGT and P-UGT mRNAs. In the other patient, a C----T substitution in exon 2 (common region) of the B-UGT/P-UGT locus resulted in a premature stop codon. This exon (132 nt) was absent in heptic B-UGT and P-UGT mRNAs of this patient due to exon skipping during pre-mRNA processing. Sequence abnormality of three distinct mRNA species explains the abnormality of multiple UGT isoforms in these patients. Presence of identical abnormalities in the common regions of the three mRNAs is consistent with the finding that the common 3' regions of the two B-UGT mRNAs and the P-UGT mRNA are encoded by four shared exons.

The expression of specific UDP-glucuronosyltransferase isoforms in 2-acetylaminofluorane—induced rat liver preneoplastic nodules was studied; livers from pair-fed littermates were used as controls. For comparison, liver and kidney from 3-methylcholanthrene—treated or untreated (control) rats were used. Steadystate UDP-glucuronosyltransferase mRNA levels were determined by Northern blot analysis or in situ hybridization of tissue sections using a 30-mer oligonucleotide specific for the 3-methylcholanthrene—inducible UDP-glucuronosyltransferase (which is active toward 4-nitrophenol) or a double-stranded cDNA probe specific for androsterone—UDP-glucuronosyltransferase. For 3-methylcholanthrene—inducible UDP-glucurono-syltransferase, the mRNA level was very low in control liver; there was a 15-fold increase after 3-methylcholanthrene treatment. This mRNA was present at relatively high concentration in the kidney and there was a threefold increase after 3-methylcholanthrene administration. In livers with preneoplastic nodules 1 mo after cessation of carcinogen administration, this mRNA concentration was approximately 15 times greater than in control liver. Similar changes in the level of the 3-methylcholanthrene—inducible UDP-glucuronosyltransferase were also observed by in situ hybridization of tissue sections. Immunocytochemical studies using an antiserum that recognizes the 3-methylcholanthrene–inducible UDP-glucuronosyltransferase showed a marked increase in the concentration of this isoform in preneoplastic nodules compared with the adjacent nonnodular liver. Consistent with the changes in mRNA levels, there was a threefold increase in 4-nitrophenol–UDP-glucuronosyltransferase activity in 3-methylcholanthrene–treated rat livers and in livers with preneoplastic nodules. In contrast to the 3-methyl-cholanthrene–inducible UDP-glucuronosyltransferase mRNA, the androsterone–UDP-glucuronosyltransferase mRNA was abundant in the liver and nearly absent in the kidney; its concentration was not increased after 3-methylcholanthrene administration or in preneoplastic nodules. Immunocytochemical studies using an antiserum that recognizes androsterone- testosterone– and bilirubin–UDP-glucuronosyltransferase isoforms did not show any increase of the concentration of these antigens in preneoplastic nodules. Similarly, activities for androsterone, testosterone and bilirubin also did not increase after 3-methylcholanthrene administration or in livers bearing the preneoplastic nodules. (HEPATOLOGY 1991;13:38—46).

Isolated cryopreserved human liver cells, attached to collagen-coated microcarriers, were injected intraperitoneally into mutant rat recipients genetically deficient in either bilirubin uridine diphosphoglucuronosyltransferase activity (Gunn rats) or albumin synthesis (Nagase analbuminemic rats). One group of the recipient Gunn and analbuminemic rats were made genetically immunodeficient by interbreeding with athymic rats with inherited T-cell deficiency. Injected microcarriers and cells formed aggregates on the surface of the pancreas. There was no morphologic evidence of rejection in athymic recipients, whereas immunocompetent recipients demonstrated rejection within 5 days of transplantation. Athymic-Gunn rat recipients demonstrated excretion of bilirubin glucuronides in bile for 30 days and reduction in their serum bilirubin levels. In recipient athymic-analbuminemic rats, plasma albumin levels increased from pretransplantation levels of 0.025–0.05 mg/ml to 3.9–4.8 mg/ml 8 days posttransplantation and remained nearly at that level throughout the 30 days of the study. A method of harvesting, attaching to microcarriers, cryopreserving, and in vivo testing of human hepatocytes with prolonged survival and function in athymic-Gunn and athymic-analbuminemic hybrid rats is reported. These rats are excellent animal models for testing xenograft function.

Adult laying domestic ducks ( Anas platyrhynchos domesticus ) were forced fed lindane (γ-hexachlorocyclohexane) 20 mg/kg body weight daily, thrice/ week and twice/week for 8 weeks. Significant shell thinning (18–21%) was observed in ducks with daily and thrice/week lindane feeding. SEM studies of thinned eggshells revealed reduced and tightly clustered mammillae with apparent decrease in intermammillary spaces and signs of inhibition of calcite formation. Such structural changes of thinned shells were associated with a significant reduction in the level of calcium both of the plasma and of shell gland of the ducks. Simultaneously, density of the pore in the shell and pore area/cm 2 of the shell in these birds were drastically reduced, presumably because of total absence of ‘large’ pores in such shells. Such porosity change is suggestive of reduced conductivity of eggshell implying impaired hatching success. Diethyl stilboestrol post-treatment neutralized all the adverse effects of lindane on duck eggshell, indicating induced estrogen deficiency in such birds by lindane.

Hematological responses of bluerock pigeon (Columba livia Gmelin) were studied after oral administration of chlordane (a cyclodiene) fenitrothion (a phosphothioate) and carbaryl (a carbamate) for one week. Comparable hematological disorders were induced by these insecticides which include reduction in total count of peripheral erythrocytes, hemoglobin content, hematocrit and total cellularity of spleen. Total count of peripheral leucocytes, on the otherhand, increased with marked heterophilia together with lymphopenia and monocytopenia. Both bleeding and clotting time became conspicuously prolonged in the experimental birds. The results indicate potential to use hematological responses for rapid on the spot assay of insecticide toxicity in non-target animals.

Lindane (γ-Hexachlorocyclohexane) was orally given to pregnant Swiss female mice at various stages of pregnancy. During early pregnancy (1–4 days of gestation), the insecticide caused total absence of any implantation site, while given during mid pregnancy (6–12 days of gestation), lindane caused total resorption of fetuses. Lindane administration during late pregnacy (14–19 days of gestation) resulted in death of all pups either within 12 h (high-dosed group) or 5 days (low-dosed group) of parturition. Body weight of such pups were also highly reduced. When estrogen was given together with lindane at early pregnancy, implantation was normal, although subsequent fetal development was adversely affected. Progesterone, unlike estrogen, could not correct lindane-induced failure in implantation. On the other hand, when estrogen and progesterone were simultaneously given to lindane-fed mice during early pregnancy, both implantation and subsequent fetal development became comparable to normal mice. The insecticide besides being fetotoxic, thus appears to cause steroid hormone deficiency resulting in reproductive and developmental failure.

Partial purification of the dihydropyridine receptor from rat skeletal muscle demonstrated mainly a 60 kDa band in SDS-polyacrylamide gel. An antibody raised against that protein behaved as a calcium channel agonist viz. Bay K8644. The affinity purified antibody, when added to cultured heart cells, increased the beat rate 40–80% depending on the titer of the antiserum. The antibody also woke up the beats of the cells previously blocked with the channel antagonist, nifedipine. Immunoblot analysis indicated that the receptor of this antibody in heart cell membrane is also a 60 kDa protein.

Lindane (gamma-hexachlorocyclohexane) was administered to laying ducks (Anas platyrhynchos domesticus) in 3 doses viz. (1) 20 mg/kg body weight daily, (2) 20 mg/kg body weight thrice weekly and (3) 20 mg/kg body weight twice weekly for 8 weeks. Egg laying was immediately stopped for 3 weeks in the first 2 groups and when resumed it became highly irregular with clutch size drastically reduced (1 against 7 of control ducks). The initial delay was absent in the 3rd group and reduction in clutch size was also marginal (5 against 7). Laying frequencies were 8.3%, 11.7% and 40% in Groups 1, 2 and 3 birds respectively against 50% frequency of the normal ducks. Hepatic, plasma and ovarian vitellogenin levels were substantially reduced in the first 2 groups. At the same time liver RNA level was markedly reduced. Ovary of such birds lack mature vitellogenic follicles and the thecal layer of moderately differentiated oocytes became highly atrophic. Stilbestrol (50 mg/kg body wt--single injection) administration to lindane fed pauci-layer ducks restored egg laying within 24 h, with return of a normal level of plasma vitellogenin and hepatic RNA. The results suggest that lindane induced estradiol insufficiency causes inhibition of hepatic RNA and yolk protein synthesis, thereby preventing transformation of moderately differentiated oocytes to mature vitellogenic follicles, delaying ovulation and thus drastically reducing clutch size.

Wistar Female rats bearing DMBA induced mammary tumours were subjected to whole body hyperthermia 42 degrees C dry heat exposure for 15 minutes daily for 6 weeks. The control group was maintained at a room temperature of 25 degrees C. Hyperthermia induced significant growth stimulation of breast tumour compared to the controls. Plasma estradiol was slightly decreased while total T4 and TSH values remained unchanged in heat stressed rats. Plasma prolactin was significantly increased together with enhanced synthetic activity of pituitary prolactin cells. It is concluded that heat acting as stressor accelerates breast tumor growth, probably by influencing synthesis of prolactin. Therefore the hormone dependency of tumours should be considered before hyperthermia is used as an anticancer modality.