Sialoadenectomy alters liver cell turn-over and function in mice

In rodents, submandibular salivary glands accumulate a number of biologically active peptides, and release some of them to both saliva and the bloodstream. Surgical removal of these glands (sialoadenectomy) alters the ability of the liver to regenerate after partial hepatectomy. We show here that 5 weeks after surgery, the liver of sialoadenectomized mice contained 40% fewer hepatocytes than the liver of sham-operated mice. We did not obtain evidence of necrotic cell death after surgery. In contrast, sialoadenectomy transiently increased apoptotic hepatocyte death, as revealed by terminal deoxynucleotidyl transferase(TdT)-mediated dUTP nick-end labeling (TUNEL) assay. DNA synthesis was determined in vivo by the incorporation of bromo-deoxyuridine (BrdU) into hepatocyte nuclei. BrdU-labeling progressively increased after sialoadenectomy. We conclude that sialoadenectomy induced a transient wave of apoptotic cell death followed by a rise in DNA synthesis but not by cell division. This reduced cell number but increased mean cell volume. In spite of these alterations in cellularity, the liver responded adequately to several stressful conditions, as judged by the lack of any differential effect of sialoadenectomy on liver glycogen and plasma glucose concentration after immobilization, aggressive encounter, or fasting. However, the liver of sialoadenectomized mice was more sensitive to the effect of a non-lethal dose of bacterial lipopolysaccharide (LPS) combined with d-galactosamine, as shown by the enhanced rise in plasma alanine aminotransferase and aspartate aminotransferase, and liver myeloperoxidase (MPO) activities. All these results indicate that a submandibular salivary glands-liver axis is involved in the maintenance of liver structure in mice. A disturbance of this axis induces an adaptive response that preserves the metabolic function of the liver but renders it more sensitive to bacterial endotoxins.     

Effect of sialoadenectomy on stomach lesions induced by indomethacin and ethanol in relation to gastric vascular permeability, the gastrin level and HCl secretion in rats.

Stomach lesions induced by indomethacin (20 mg.kg-1 i.p.) and ethanol (1 ml 95% intragastrically) were studied after a 24 hour fast in rats which had undergone sialoadenectomy. The size of the lesions was correlated with gastric HCl secretion, with gastric vascular permeability (determined from the Evans blue concentration in the stomach tissue after its i.v. administration) and with the serum gastrin level. These parameters were also studied in sialoadenectomized rats and in animals given epidermal growth factor (EGF) (50 lg.kg-1). It was found that sialoadenectomy significantly (p < 0.01) raised the incidence of stomach lesions after the administration of indomethacin and also after ethanol (p < 0.05). A significant increase in both basal and stimulated HCl secretion was found after sialoadenectomy. Both indomethacin and ethanol also increased gastric vascular permeability in rats not subjected to sialoadenectomy, but sialoadenectomy raised it significantly compared with the non-sialoadenectomized group. The serum gastrin levels fell after sialoadenectomy and the decrease was significant after the subsequent administration of indomethacin or ethanol. The administration of EGF to sialoadenectomized rats lowered the incidence of stomach lesions, inhibited HCl secretion and reduced vascular permeability. The lowered susceptibility of the gastric mucosa to the formation of lesions in sialoadenectomized rats given indomethacin or ethanol can be regarded as the outcome of the uptake of EGF.     

Submandibular salivary glands: influence on growth rate and life span in mice

Submandibular glands accumulate a variety of growth factors, especially in male mice. Surgical excision of these glands (sialoadenectomy) results in alterations in several organs and systems including the liver, skin and reproductive system. We studied the life-long consequences of sialoadenectomy in male mice. Animals were operated at the age of 10 weeks. Thereafter, body weight and food and water intake were controlled until death. Few weeks after surgery, body weight was lower in sialoadenectomized than in control mice. The difference remained stable until the age of 80 weeks. In spite of the lower body weight, food intake was higher in sialoadenectomized mice than in controls. The first death of sialoadenectomized mice occurred 10 weeks earlier than that of the first control, and the initial death rate in sialoadenectomized mice was almost twice the rate in controls. After 100 weeks of life, the death rate increased in control mice, but suddenly decreased in sialoadenectomized mice. The consequence was that the mean life span of the last 25% surviving animals was 10 weeks longer in sialoadenectomized than in control mice. Autopsy examination suggests that the effect of sialoadenectomy on death rate may be the consequence of a contrasting effect on tumour growth. Our results indicate that submandibular glands, or rather the factors derived from these glands, have contrasting roles in tumour growth. At early ages they may be survival factors and protect tissues, whereas at later ages they may stimulate the growth of transformed cells.     

Influence of submandibular salivary glands on hormone responsiveness of mouse mammary glands

Surgical removal of the submandibular salivary glands (sialoadenectomy) of female Balb/c mice significantly (P less than 0.05) reduced mammary development as judged by development scores and mammae DNA levels. Reduction in mammae development score by sialoadenectomy was observed in both mice saline injected and mice treated with estradiol and progesterone. Autografts of submandibular salivary tissue or daily administration of EGF to sialoadenectomized mice partly alleviated the atrophy of the mammary gland induced by sialoadenectomy (P less than 0.05). The results of our studies are consistent with a model of mammary gland developmental regulation that includes the submandibular salivary gland as a mediator of mammogenesis via secretion of EGF.     

Verhulstian analysis of the growth of transplantable mammary tumours in sialoadenectomized mice

In this report we have analysed data published in 1989 by Inui et al. (Incidence of precancerous foci of mammary glands and growth rate of transplantable mammary cancers in sialoadenectomized mice. J. Natl Cancer Inst. 81, 1660) involving the effects of perturbation of the epidermal growth factor (EGF) status of mammary tumour-bearing mice on subsequent volumetric responses. Removal of an endogenous EGF stimulus by surgical ablation of the submaxillary glands, the major EGF-producing organ in mice, produced significantly slower growth of rodent mammary neoplasms, decreased success rate of transplantation, and an increase in the latent period before growth occurred. Administration of i.p. EGF (5 micrograms/mouse/day) to sialadectomized tumour-bearing mice would however, increase tumour growth rate. Data were analysed using the Verhulst equation which indicated that the observed effects on tumour volumetrics by either sialoadenectomy or EGF administration could be interpreted as being produced through paracrine pathways. The use of the Verhulstian analysis indicates that it is possible to analyse neoplastic responses and infer whether paracrine or autocrine pathways are involved.     

The role of macrophages in the early resistance to mouse hepatitus virus infection in nude mice.

Nude mice which had received intraperitoneal injection of silica simultaneously with infection of mouse hepatitis virus, NuU strain, died of severe necrotic hepatitis within 2 weeks postinfection, whereas those having received no silica survived for 3 weeks or more after challenge. Silica given day 4 postinoculation had no effect. The virus titers of the liver and spleen at day 4 as well as serum interferon levels at day 2 were much higher in silica-treated mice than those without silica treatment. At day 2 or 3 postinoculation, silica-treated mice were found to have a considerable number of necrotic foci in the liver with some neutrophil and lymphocyte infiltration, and viral antigen was present in the cytoplasm of some hepatocytes around necrotic foci. In contrast, those without silica treatment showed only some necrotic foci with some lymphocyte infiltration. Viral antigen was detected only in a few littoral cells but not in hepatocytes. The role of macrophages in the resistance at early stage of inection in nude mice is discussed.     

The effect of submandibular gland removal on testicular and epididymal parameters

The effect of sialoadenectomy (submandibular gland removal) for four weeks duration was studied in five C3H mice and four sham-operated controls. This preliminary experiment using small numbers of animals indicated that sperm production parameters were lowered in sialoadenectomized animals although there were no significant differences between the sialodenectomized and sham-operated group. In a second experiment using larger numbers of animals, significant differences in testicular weights, epididymal weights, and testis weights were noted. These parameters were reduced only 10-14% from control levels. Epididymal sperm head counts were reduced 10% after sialoadenectomy but the two groups were not significantly different (P less than 0.05). No difference in testicular histology was observed. The present report contrasts to that of Tsutsumi et al. (1986) who indicate that submandibular removal with subsequent depletion of epidermal growth factor (EGF), is extremely deleterious (40-55% decreases in spermatid and sperm content) to the progress spermatogenesis.     

Immobilization stress induces c-Fos accumulation in liver

Acute stress-induced injury in tissues has been revealed by both biochemical markers in plasma and microscopy. However, little is known of the mechanisms by which tissue integrity is restored. Recently, induction of early response genes such as c-fos has been reported in the heart and stomach of immobilized animals. Herein, we show that immobilization stress in mice increased plasma alanine aminotransferase activity, a marker of liver damage. c-Fos protein accumulation in liver was induced by stress after 20 minutes of immobilization and persisted for 3 hours. Immobilization also induced the release of epidermal growth factor (EGF) from submandibular salivary glands and a transient increase in EGF concentration in plasma. Although EGF administration induced a 2.5-fold increase in c-Fos mass in the liver of anesthetized mice, sialoadenectomy (which abolished the effect of immobilization on plasma EGF) did not affect the stress-induced rise in plasma alanine aminotransferase activity or liver c-Fos accumulation. Therefore, we conclude that immobilization stress induces c-Fos accumulation in liver and that this effect is not triggered by the increase in plasma EGF concentration.     

Liver injury after an aggressive encounter in male mice

Acute and intense psychological stressors induce cell damage in several organs, including the heart and the liver. Much less is known about social stress. In male mice, aggressive behavior is the most common social stressor. It is remarkable that upon fighting, submandibular salivary glands release a number of peptides into the bloodstream including epidermal growth factor (EGF). We showed previously that released EGF protects the heart from cell damage in this particular stressful situation. Here, we studied the effect of an aggressive encounter on the liver and whether EGF has a similar effect on this organ. An aggressive encounter in male mice caused inflammatory response and a transient increase in plasma alanine and aspartate transaminase activities. At 3 h, focal infiltration of neutrophils was observed in liver parenchyma. These cells accumulate on eosinophilic hepatocytes, which may correspond to dying cells. A few hours later, evidence of necrotic lesion was observed. Surgical excision of submandibular glands, sialoadenectomy, did not prevent the rise in plasma EGF concentration and did not affect the increase in plasma transaminase activities. Neither did the administration of tyrphostin AG-1478 (inhibitor of EGF receptor kinase) alter the increase in plasma alanine transaminase activity. However, it did enhance the rise in both aspartate transaminase and creatine kinase activity, suggesting heart damage. We conclude that an aggressive encounter causes mild liver damage and that released EGF does not protect this organ, in contrast to its effect on the heart.     

The effects of sialoadenectomy and exogenous EGF on taste bud morphology and maintenance

Taste buds on the dorsal tongue surface are continually bathedin saliva rich in epidermal growth factor (EGF). In the followingexperiment, taste bud number and morphology were monitored followingsubmandibular and sublingual salivary gland removal (sialoadenectomy),to determine if EGF plays a role in the maintenance and formationof taste buds. Adult male rats were divided into four groups:sialoadenectomized (SX, n = 4); sialoadenectomized with EGFreplacement (SX + EGF, n = 5); sham-operated (SH, n = 4); andsham-operated with exogenous EGF (SH + EGF, n = 5). After a3 week recovery, SX + EGF and SH + EGF animals were given 50µg/day EGF in their drinking water for 14 days. At day14, saliva was collected, the animals were killed and the presenceof EGF determined by radioligand-binding assay. Tongues wereremoved and histologically examined for the presence and morphologyof taste buds on fungiform and circumvallate papillae, or immunostainedfor the presence of EGF, TGF     

Chronic ethanol exposure potentiates lipopolysaccharide liver injury despite inhibiting Jun N-terminal kinase and caspase 3 activation.

Although ethanol is known to sensitize hepatocytes to tumor necrosis factor (TNF) lethality, the mechanisms involved remain controversial. Recently, others have shown that adding TNFalpha to cultures of ethanol-pretreated hepatocytes provokes the mitochondrial permeability transition, cytochrome c release, procaspase 3 activation, and apoptosis. Although this demonstrates that ethanol can sensitize hepatocytes to TNF-mediated apoptosis, the hepatic inflammation and ballooning hepatocyte degeneration that typify alcohol-induced liver injury suggest that other mechanisms might predominate in vivo. To evaluate this possibility, acute responses to lipopolysaccharide (LPS), a potent inducer of TNFalpha, were compared in mice that had been fed either an ethanol-containing or control diet for 5 weeks. Despite enhanced induction of cytokines such as interleukin (IL)-10, IL-15, and IL-6 that protect hepatocytes from apoptosis, ethanol-fed mice exhibited a 4-5-fold increase in serum alanine aminotransferase after LPS, confirming increased liver injury. Six h post-LPS histology also differed notably in the two groups, with control livers demonstrating only scattered apoptotic hepatocytes, whereas ethanol-exposed livers had large foci of ballooned hepatocytes, inflammation, and scattered hemorrhage. No caspase 3 activity was noted during the initial 6 h after LPS in ethanol-fed mice, but this tripled by 1.5 h after LPS in controls. Procaspase 8 cleavage and activity of the apoptosis-associated kinase, Jun N-terminal kinase, were also greater in controls. In contrast, ethanol exposure did not inhibit activation of cytoprotective mitogen-activated protein kinases and AKT or attenuate induction of the anti-apoptotic factors NF-kappaB and inducible nitric oxide synthase. Consistent with these responses, neither cytochrome c release, an early apoptotic response, nor hepatic oligonucleosomal DNA fragmentation, the ultimate consequence of apoptosis, was increased by ethanol. Thus, ethanol exacerbates TNF-related hepatotoxicity in vivo without enhancing caspase 3-dependent apoptosis.     

Effects of sialoadenectomy and epidermal growth factor administration on the duodenum of adult mice.

Sialoadenectomy (removal of the submandibular glands) of adult male and female mice caused significant or apparent decreases in duodenal weight, duodenal protein, DNA and RNA contents, and alkaline phosphatase activity. These decreased levels observed in sialoadenectomized mice were completely restored to or rather increased over the control levels by epidermal growth factor (EGF) administration. There was no appreciable difference in body weight between the control and sialoadenectomized mice. These results strongly suggest that salivary EGF secreted from the submandibular gland plays a role in the maintenance of growth and function(s) of the adult mouse duodenum.     

A strong genotoxic effect in mouse skin of a single painting of coal tar in hairless mice and in MutaMouse

The dorsal skin of C3H/Tif/hr hairless mice was painted with coal tar, pharmacological grade. Epidermal cells and hepatocytes were isolated after 4, 24, 48 and 96 h and DNA strand breaks were determined as tail moment by the alkaline comet assay. The tail moment of epidermal cells was significantly greater at the time points 4, 24, 48 and 96 h after exposure compared to the controls, with the most DNA strand breaks at 24 h. The DNA strand breaks in epidermal cells increased linearly with the dose of coal tar. In hepatocytes, no difference in DNA strand breaks was found between exposed animals and controls. DNA adducts were determined by the 32P-postlabeling assay. For epidermal cells, the mean DNA adduct level was 12-fold greater in coal tar painted mice after 24 h than in controls. Again, a linear dose/response relationship was seen 24 h after painting. For liver DNA, the mean DNA adduct level was 3-fold greater than for controls. The mutation frequency in epidermal and liver cells was examined in lambdalacZ transgenic mice (MutaMouse). Thirty-two days after painting, the mutation frequency in epidermal cells was 16-fold greater in coal tar treated mice compared to controls. No effect was detected in hepatocytes. We found that a single painting of coal tar resulted in strong genotoxic effects in the murine epidermis, evidenced by induction of DNA strand breaks and DNA adducts in hairless mice and lambdalacZ mutations in the MutaMouse. This demonstrates that it is possible to detect genotoxic effects of mixtures with high sensitivity in mouse skin by these end-points.     

EGF-Induced receptor autophosphorylation in primary hepatocytes isolated from phenobarbitone-treated mice.

Phenobarbitone (PB) treatment of mice causes a decrease in the growth factor responsiveness of hepatocytes. Here, epidermal growth factor receptor (EGFR) expression and receptor autophosphorylation was determined in hepatocytes isolated from control and PB-treated mice. There was a decrease in the level of EGFR expression in hepatocytes isolated from mice following PB administration when compared to controls. EGF caused an approximate 20-fold increase of the 170 kD phosphotyrosine band in control hepatocytes, which was inhibited by the EGFR specific tyrosine kinase inhibitor 4, 5-dianilinopthalamide. Following PB treatment, the degree of basal receptor phosphorylation (in the absence of EGF) was significantly greater and therefore the fold rise in EGFR phosphorylation in isolated hepatocytes was lower than in controls. However, the overall extent of EGF-induced receptor phosphorylation was not diminished in hepatocytes isolated from PB-treated mice. Therefore the reduction in responsiveness to growth factors seen in hepatocytes ex vivo or the cessation of proliferation observed in vivo following PB administration is unlikely to be attributed to a decrease in ligand binding and subsequent receptor autophosphorylation.     

Effects of estradiol and progesterone on epidermal growth factor concentration in plasma, bile, urine, submandibular gland and kidney of the mouse

Testosterone is known to increase epidermal growth factor (EGF) concentrations in mouse plasma and submandibular salivary gland. We tested in adult sialoadenectomized (sx) and sham-operated female and male mice our hypothesis that female sex steroids also affect EGF concentrations in fluids and tissues. In 10-day treatment estradiol-17 beta increased the EGF concentration in male urine and in (sx) female plasma. Progesterone increased the concentration in both sexes in plasma (sx mice) and in the kidneys. In contrast, progesterone decreased it in female urine.     

Effect of sialoadenectomy on ethanol-induced gastric mucosal damage in the rat: role of neutrophils

We have observed that removal of the salivary glands is associated with an increase in the susceptibility to gastric mucosal damage in the rat. In the present study, we have examined the effect of sialoadenectomy on ethanol-induced mucosal hemorrhagic damage and myeloperoxidase (MPO) activity. Hemorrhagic damage and MPO activity in response to intragastric 50% w/v ethanol were greater in sialoadenectomized rats when compared with sham-operated animals. Pretreatment with 16,16-dimethylprostaglandin E2 (0.3 micrograms/kg s.c.) reduced damage and MPO activity in both sialoadenectomized and sham control rats receiving 50% ethanol. The reduction in these parameters was greater in control than in sialoadenectomized rats. Pretreatment with epidermal growth factor (5 micrograms/kg s.c.) significantly reduced MPO activity but did not significantly affect the extent of damage. These data suggest that sialoadenectomy is associated with an increase in mucosal inflammation in animals given ethanol. However, in some situations tissue inflammation (as indicated by MPO activity) was reduced, while the proportion of gastric mucosa exhibiting hemorrhagic damage was not changed.     

The influence of sialoadenectomy, thymectomy and starvation on liver glycogen in the rat

Hepatic glycogen was assayed in young and adult rats subjected to sialoadenectomy and/or thymectomy and starvation. Sialoadenectomy in young, but not in adult rats caused the rats to stop feeding. In young, but not in adult sialoadenectomized and starved rats the glycogen level was notably higher than in unoperated and starved rats, indicating active participation of salivary glucagon in immature animals in hepatic glycogenolysis under conditions of starvation. Simultaneous sialoadenectomy and thymectomy caused glycogen depletion in the liver of young rats in spite of the absence of the salivary glands. Acceleration of glycogenolysis in these rats was not due to thymectomy, being probably a result of excessive secretion of adrenal catecholamines.     

The calcium-dependence of the stimulation of neonatal rat hepatocyte DNA synthesis and division by epidermal growth factor, glucagon and insulin

A low concentration (10(-11) mol/l) of epidermal growth factor (EGF) and/or an equimolar (10(-14) mol/l) mixture of glucagon and insulin stimulated DNA synthesis in hepatocytes in 4-day-old primary cultures of neonatal rat liver. EGF seems to have acted by inducing quiescent hepatocytes to begin cycling, while the glucagon-insulin combination seems to have acted mainly by shortening the cell cycle time. Incubation in low calcium medium blocked untreated hepatocytes in the G1 phase of their cycle and prevented EGF and the glucagon-insulin mixture from stimulating DNA synthesis. Nevertheless, hepatocytes in calcium-deficient medium did respond to these agents, as they reached a late stage of prereplicative development before being blocked: in fact, they initiated DNA synthesis soon after the addition of calcium. EGF, but not the glucagon-insulin combination, also enabled the already cycling hepatocytes (but not the newly activated ones) to overcome the block imposed by the extracellular calcium deficiency after a delay of several hours.