A study of tobacco carcinogenesis, LIII: carcinogenicity of N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in mink (Mustela vison).

In an earlier study, young male and female mink (Mustela vison) were found to be highly susceptible to the carcinogenic effect of N'-nitrosonornicotine (NNN). In this follow-up study we tested (i) the importance of the age of the animals with regard to the carcinogenic effect of NNN, (ii) the carcinogenic activity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and (iii) the combined carcinogenic effect of NNN plus NNK. (I) In the previous study, the latency of nasal tumor induction by NNN (11.9 mM) averaged 84 +/- 40 weeks upon twice weekly applications, starting at the age of 3 weeks and continuing for 38 weeks. In this bioassay, giving NNN in 28 weeks but starting at the age of 3 months, it took, on the average, 97 +/- 29 weeks to induce malignant nasal tumors, primarily esthesioneuroepithelioma with invasion of the brain. (ii) NNK (6.3 mM), given by s.c. injection, induced nasal carcinoma with invasion of the forebrain after 77 +/- 39 weeks. (iii) NNN (11.9 mM) plus NNK (6.3 mM) led to the same type of carcinoma but at an accelerated pace, namely after 71 +/- 57 weeks. This study supports the earlier observation that tobacco-specific N-nitrosamines induce malignant tumors of the nasal cavity with invasion of the brain, dependent to some degree on the age of the mink at first application. NNK appears to be a stronger carcinogen than NNN in mink which follows the observations made with mice, rats and hamsters. It is suggested that combined administration of NNN with NNK induces a stronger carcinogenic effect than NNN or NNK given alone.

Decline in glucose metabolism in the brain in neuronal ceroid lipofuscinosis (NCL) in English setter--evidence by positron emission tomography (PET).

Positron emission tomography scans were performed on brains of homozygous and heterozygous English setters with neuronal ceroid-lipofuscinosis (NCL) from 13 months of age to 24-25 months of age for homozygous dogs and to 38 months for heterozygous dogs, respectively. After iv injection of 18F-fluorodeoxyglucose (FDG), 7 coronal brain scans as well as sequential arterial blood samplings were performed for 45 min under pentobarbital anesthesia. From these data, three different functional images (DAR (standardized uptake value), FDG uptake (fractional uptake) and glucose uptake) were reconstructed and quantitatively analysed. In the age range from 13 to 15 months, glucose images were comparable for both homozygous and heterozygous dogs, so that no differentiation was possible between healthy and diseased dogs on the basis of PET findings. Between 18 and 24 months of age, a drastic decline in glucose metabolism was observed in homozygous dogs, while the decline in glucose utilization was very mild in this period for heterozygous dogs. Furthermore, in PET scans, cerebral atrophy and ventricular enlargement were clearly shown in homozygous dogs. Consequently at the age from 20 to 24 months, a clear differential diagnosis between healthy (heterozygous) and diseased (homozygous) dogs became possible even if the clinical symptoms were still not clear in the latter. We conclude that the biochemical alterations in the brain in canine NCL occurs and progresses very rapidly in the last quarter of their lives.

Lysine methylation of mitochondrial ATP synthase subunit c stored in tissues of dogs with hereditary ceroid lipofuscinosis.

Certain forms of ceroid lipofuscinosis, a hereditary degenerative disease, are characterized by accumulation of large amounts of subunit c of mitochondrial ATP synthase in lysosomal storage bodies of numerous tissues. The subunit c protein appears to constitute a major fraction of the total storage body protein. In previous studies it was demonstrated that hydrolysates of total storage body protein from affected humans and sheep contain significant amounts of epsilon-N-trimethyllysine (TML). This finding suggested that one or both of the two lysine residues of subunit c might be methylated in the stored form of the protein. The normal subunit c protein from mitochondria does not appear to be methylated. Using a putative canine model for the juvenile form of ceroid lipofuscinosis, analyses were conducted to determine whether lysosomal storage of subunit c was accompanied by lysine methylation of this protein. In affected dogs, as in humans and sheep with hereditary ceroid lipofuscinosis, the storage bodies were found to contain large amounts of subunit c protein, as indicated by polyacrylamide gel electrophoresis and partial amino acid sequence analysis. The subunit c protein partially purified from isolated storage bodies was found to contain lysine and TML in an almost equimolar ratio. Normal subunit c contains 2 lysine residues, one at position 7 and the other at position 43. Removal of the first 7 residues of the partially purified protein through sequential Edman degradation resulted in a dramatic increase in the TML to lysine ratio in the residual protein. This suggests that lysine residue 43 is methylated. Confirmation that residue 43 of the stored protein is TML was obtained by amino acid sequence analysis after cleavage of the protein with trypsin. This finding strongly suggests that specific methylation of lysine residue 43 of mitochondrial ATP synthase plays a central role in the lysosomal storage of this protein.

A study of tobacco carcinogenesis XLVIII. Carcinogenicity of N'-nitrosonornicotine in mink (Mustela vison).

During tobacco processing and smoking, nicotine and nornicotine give rise to N'-nitrosonornicotine (NNN), a highly abundant, strong carcinogen. NNN is known to exert carcinogenic activity in mice, rats and hamsters. Major target organs for NNN carcinogenicity in the rat are the esophagus and the nasal mucosa, and in the Syrian golden hamster trachea and nasal mucosa. In comparison with the rat, the mink (Mustela vison) has a markedly expanded nasal mucosa. Therefore, we explored in this study whether the mink could serve as a non-rodent model for nasal carcinogenesis using NNN as the carcinogen. Twenty random-bred mink, beginning at the age of 3 weeks, received twice weekly s.c. injections of NNN, a total dose of 11.9 mM per animal over a 38 week period. All of the 19 mink at risk developed malignant tumors of both the respiratory and the olfactory region of the nose within 3.5 years. In most animals the malignant tumors, primarily esthesioneuroepithelioma, invaded the brain. Remarkably, NNN induced no other tumors in the mink. None of the control animals developed nasal tumors nor tumors at other sites during the 3.5 years of the assay. The historical data from the farm did not reveal any spontaneous occurrence of nasal tumors in mink at any age. This study supports the concept that NNN is a proven carcinogen for multiple species of mammals and that the mink can serve as a non-rodent, non-inbred animal model for nasal carcinogenesis, especially since NNN induces only tumors in the nasal cavity in this species and not at other sites, as it does in mice, rats and hamsters.

English setter model and juvenile ceroid-lipofuscinosis in man.

The etiology of the juvenile type of the human ceroid-lipofuscinosis (JCL) is unknown, in spite of the fact that the first report of this disease was given more than 160 years ago. The necessity of good animal models for scientific progress in chronic metabolic diseases in humans is obvious. The inbred strain of English setter with ceroid-lipofuscinosis (CCL) seems to be a perfect model for human JCL. Dogs with CCL and organs for research purposes are available from Dr. Koppang's experimental kennel in Norway.

Batten's disease: failure of allogeneic bone marrow transplantation to arrest disease progression in a canine model.

We investigated whether the course of canine ceroid lipofuscinosis (CCL), a model of Batten's disease in man, was affected by allogeneic bone marrow transplantation. Four English setters with CCL, 4 1/2 months of age, were given 9.2 Gy of total body irradiation, followed by the infusion of bone marrow cells from healthy DLA identical sibling donors. All transplanted dogs had complete hematologic reconstitution. However, at 12-13 months posttransplant, all dogs developed characteristic and progressive signs of CCL. Autopsies revealed cerebral atrophy and findings of ceroid storage not different from those in non-transplanted controls. These findings suggest that bone marrow cells do not contain or release the gene product(s) necessary to correct the disease. It appears unlikely that with our current knowledge, allogeneic marrow transplantation would be beneficial in the treatment of Batten's disease.

Lectin histochemical study of lipopigments: results with concanavalin A.

Concanavalin A (Con A) binding to lipopigments (LPs) of the lipofuscin type was proved to be due to the high content of mannose. Two mannose bearing compounds could be recognized due to their different organic solvent solubility. One was best soluble in modified chloroform-methanol-water mixture (10:10:3) and corresponded most probably to the oligosaccharyl disphosphodolichol (oligo-PP-Dol) described to be significantly increased in LPs of inherited type. The second one, organic solvent insoluble corresponded to a glycoprotein (GP). The ratio of the two components varied. The deposition of the typical lipofuscin (age pigment) was dominated by the GP component. Its amount was greatest in neurolipofuscin (especially in the olivary nucleus) but very little in hepatocytic lipofuscin. In human neuronal ceroid lipofuscinoses (of early juvenile, and juvenile types) both components were found in large quantities in the storage granules of the affected neurons. The "protein type variant" of the storage material (Elleder, 1978) displayed the highest degree of lipid-bound mannose accumulation, the GP component being absent. In the late infantile, infantile and Kufs variants studied in paraffin sections only, the GP component was detectable, too as in the case of the secondary neuronal LP in mucopolysaccharidoses and gangliosidoses. In the canine model of NCL lipid bound mannose clearly predominated, the GP component being in low amount on average. Neither of the Con A reactive glycoconjugates could be identified as the component responsible for autofluorescence. However, both are most probably responsible for PAS positivity of lipofuscins. There were no detectable Con A reactive glycoconjugates in the histiocytic ceroid.

The English setter with ceroid-lipofuscinosis: a suitable model for the juvenile type of ceroid-lipofuscinosis in humans.

Genetic and histological examinations (light and EM) of tissues of an inbred line of English setters have proved that these dogs suffer a general metabolic autosomal recessive disease, canine ceroid-lipofuscinosis (CCL) almost identical to the human Stengel-Batten-Spielmeyer-Vogt disease, or neuronal ceroid-lipofuscinosis (NCL). A controlled longitudinal morphologic study showed that the formation and accumulation of an autofluorescent lipopigment, identified as "ceroid" in the isolated state, appears in the neurons already in 2-day-old puppies and increases linearly with time. Clinical signs and symptoms develop after a distinct loss of neurocytoplasm and its functional organelles is demonstrable. At that time, loss of functional neuronal cytoplasm appears to result from pigment formation. Nerve cells which have suffered this fate will eventually die and disappear. The process leads to severe global neurologic disturbance and cerebral atrophy. By the time of death from the disease at age 20 to 27 months, brain weight is reduced to 60-70% of normal control animals. The English setter with CCL differs somewhat from humans in the degree of morphological damage to various layers of the retina. In human with NCL, there is a pronounced loss of photoreceptors in the end-stage of the disease, but in CCL only minimal structural damage is observed in the retina. For experimental treatment protocols for NCL, the CCL setter is a useful model.

4-Hydroxynonenal: a specific indicator for canine neuronal-retinal ceroidosis.

Previous attempts to demonstrate abnormalities in lipid peroxidation in various forms of the neuronal ceroid-lipofuscinoses (NCL) have been unrewarding up to and including the peroxide level (peroxidase). In this experiment a survey was made in a canine model of NCL to study the relative concentration of 4-hydroxynonenal (HNE), a fragment derived from an acute oxidation product of unsaturated fatty acids. Peripheral blood cells and various tissues from an affected and a normal control dog were surveyed. HNE was assayed after reacting with O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine to form the 4-hydroxynonenal (O-pentafluorobenzyl) oxime. This reaction product was then separated by capillary gas liquid chromatography (g/c) and quantitated by flame ionization. The survey showed that neutrophils isolated from affected dogs and carriers contained abnormal amounts of HNE when compared with normal control animals. Two carriers had mean values of +3,289% above normal, and neutrophils from two affected animals were +4,873% above normal. In addition, an examination of the relative HNE levels in brain, retina, retinal pigment epithelium (RPE), and kidney of an affected dog compared with a control animal also showed abnormal levels of HNE, particularly in brain (+168%) and in the RPE (+135%), the two organs exhibiting the most severe pathologic damage unique to these disorders. These findings, although preliminary, clearly document a role for HNE in this canine form of human NCLs. The well-known cytotoxic properties of HNE and other alpha,beta unsaturated aldehydes suggest a primary role in the pathogenetic events of this disorder.

Characterization of disease-specific brain fluorophores in ceroid-lipofuscinosis.

By isolating and identifying the molecular components of the storage material in the ceroid-lipofuscinoses, it should be possible to elucidate the metabolic basis for these diseases. Using brains of English setter dogs afflicted with a form of this disorder, the autofluorescent storage granules have been isolated and subjected to extraction with chloroform-methanol. A significant amount of autofluorescent material was solubilized by this procedure. Spectral analysis of the extracts indicated that the disease-specific fluorophores have corrected fluorescence excitation maxima at 335-340 nm and emission maxima at 530 nm. Analysis of the extracts with thin layer chromatography showed the presence of several disease-related fluorophores. In addition, the amounts of several nonfluorescent lipids appeared to be enhanced as a result of ceroid-lipofuscinosis. A cerebral cortex sample from a human subject who died with the late infantile form of ceroid-lipofuscinosis was subjected to the same analytical procedures. The chloroform-methanol extract from the human brain had a fluorescence spectrum very similar to the extracts from the brains of affected dogs. TLC analysis showed a single fluorophore from the human brain sample. This fluorophore had the same retention time as the most prominent fluorophore present in the samples from affected dogs. These findings are consistent with the possibility that the defects underlying the human and canine ceroid-lipofuscinoses are similar. The analytical procedures established in these experiments are useful in evaluating the various models of ceroid-lipofuscinosis and may ultimately serve as the basis for distinguishing among different forms of these disorders.(ABSTRACT TRUNCATED AT 250 WORDS)

Vascular changes and liver tumours induced in mink by high levels of nitrite in feed.

Two groups of female mink were fed a diet supplemented with 30-50 mg/kg bw sodium nitrite for up to six years. The first group also received dimethylamine hydrochloride. Seven male offspring from litters born in the first year were fed the same diet for nine months but showed no pathomorphological change. After three years on trial, female mink developed occlusive changes in some branches of the efferent hepatic veins, and 21% of the mink in group 1 and 31% in group 2 developed liver haemangioendotheliomas or precancerous liver changes. The pathomorphological changes were identical to those seen in animals exposed to N-nitrosodimethylamine (NDMA). This result indicates in-vivo formation of NDMA as a result of the high nitrite in the diet. However, NDMA was not measured in the blood of the nitrite-exposed mink.

Ultrastructural study of primary canine and human pigmentary retinopathy.

An electron microscopic study was performed on eyes of Labrador dogs afflicted with progressive retinal atrophy (PRA). There was complete loss of photoreceptors, atrophy of the remaining retina and gliosis in the peripheral part while the central retina showed incomplete loss of photoreceptors and an almost total disappearance of photoreceptor outer segments. Melanin-bearing cells, largely containing melanolysosomes, were found deep inside the retina. This electron microscopic study also incorporated the retina of a middle-aged woman affected by retinopathia pigmentosa (RP). The fine structure of the diseased retina showed a similar pattern of lesions, more pronounced in the periphery of the retina. Similar electron microscopic findings between the two disease processes render PRA of the Labrador dog a useful model for a comparative study of the development and intraretinal spread of human RP.

Dolichol and dolichyl phosphate levels in brain tissue from English setters with ceroid lipofuscinosis.

Human ceroid lipofuscinosis (CL) is an inherited disease marked by cerebromacular degeneration and early death. We have utilized the canine model to investigate the possible role of dolichol and dolichyl phosphate in the developmental pathology of CL. We found that while brain levels of dolichol increase with age in both affected and unaffected dogs, the amount in the diseased animal was similar to that in controls. Brain levels of dolichyl phosphate ranged from 20 to 35 micrograms/g in control dogs at all ages examined, but increased substantially during development in the affected dogs, a value of 113 +/- 24 micrograms/g (mean +/- SD) being obtained in the end-stage animals. In addition to the results obtained in the canine model, dolichyl phosphate levels in human brain tissues from a 5-year-old with late infantile CL and from a 19-year-old with juvenile CL were found to be 153 and 382 micrograms/g, respectively, compared with a control that assayed 26 micrograms/g. The preliminary findings with human tissues provide further evidence for an association of elevated brain dolichyl phosphate levels with CL. Whether the increase is primary or secondary remains to be determined.

Canine ceroid lipofuscinosis, a model for ageing of the human isocortex.

In canine ceroid lipofuscinosis (one case studied), isocortical layer IIIab pyramidal cells develop spindle-shaped enlargements of their proximal axon filled with lipopigment, a feature that can be observed in juvenile and adult type of human neuronal ceroid lipofuscinosis and in normal ageing of the human isocortex as well.

Studies on the retina and the pigment epithelium in hereditary canine ceroid lipofuscinosis. IV. Changes in the electroretinogram and the standing potential of the eye.

The present study correlates ultrastructural abnormalities in the retinal pigment epithelium (RPE) with electrophysiologic changes demonstrated in examinations of dogs with canine ceroid-lipofuscinosis (CCL) at various stages of clinical involvement using dc electroretinograms (ERG), including the c-wave, as well as recordings of the variations of the standing potential (SP) of the eye, both of which reflect the activity of the pigment epithelium. Normal c-waves and SP variations were seen at early stages of disease. At later stages, specific signs of pigment epithelial impairment were found in the form of (1) markedly reduced SP variations, (2) an increase in amplitude of the trough after the b-wave, and (3) a disappearance of the c-wave, which was replaced by a negative potential. These ERG changes correlate very well with a damaged pigment epithelium that generates the positive potential of the c-wave, as well as with normal Müller cells that generates the negative potential coinciding in time with the c-wave, and contributing to the trough after the b-wave. In the end-stage of the disease all potentials of the ERG were reduced drastically, evidently as a sign of cessation of neuroretinal function. Similarities between English setters with CCL and human Batten disease are discussed.

Computed tomographic and nuclear magnetic resonance correlation of canine ceroid-lipofuscinosis with aging.

Canine ceroid lipofuscinosis is a degenerative neuronal disorder which has as a distinct pathologic counterpart, neuronal ceroid lipofuscinosis or Batten's disease in humans. The disease occurs in English setters and is associated with abnormal accumulation of autofluorescent lipopigments. As such, a study of this animal model may allow insight into the aging process as well. This investigation explores the computed tomographic features of canine ceroid-lipofuscinosis and correlates these findings (progressive atrophy, inherent changes in CT/NMR tissue characteristics) with clinical and pathologic features.

Canine hereditary ceroid lipofuscinosis.

Dogs with an inherited form of ceroid lipofuscinosis are ataxic, blind and demented. During the disease process, they undergo severe cerebrocerebellar atrophy with storage of autofluorescent, lipid peroxide-positive reacting substances whose ultrastructure resembles 'fingerprint' patterns of membranous lamellae. The retina and RPE also undergo pathologic changes. Most important is the inverse relationship between loss of RPE melanin and increased deposition of ceroid. These pathological events in brain and eye lead to altered EEG, ERG and VEP activity. This inbred strain of English setters fulfills essentially all the criteria as a model for the human disease and will prove useful in the future for therapeutic trials.