The prevention of fatalities in cycloheximide challenged mice pretreated with a submicrogram dose of lipopolysaccharide or with a small volume of cell-free Ehrlich ascites tumour fluid.

OBJECTIVE AND DESIGN: Therapeutic strategies were employed to better understand the pathogenesis of fatal cycloheximide (CX) intolerance in mice pretreated with a submicrogram dose of lipopolysaccharide (LPS) or with a small volume of cell-free Ehrlich ascites tumour fluid (EAF). MATERIALS AND SUBJECTS: Inbred, male CBA-strain mice. METHODS: Aminoguanidine (AMG), a selective inhibitor of inducible nitric oxide synthase (iNOS) was used to determine the role of nitric oxide (NO) in the fatalities. Dexamethasone (DEX) or nordihydroguaiaretic acid (NDGA) were used to assess possible involvement of tumour necrosis factor-alpha (TNF) in sensitisation to CX. RESULTS: AMG protected CX challenged mice pretreated with LPS or with EAF. DEX, but not NDGA, protected the former animals; both DEX and NDGA protected the latter. CONCLUSION: AMG protection indicates the essential role of iNOS in CX-precipitated fatalities. It is suggested that the pretreatments either directly or indirectly place iNOS expression under control of a labile protein repressor. In the case of EAF pretreated mice, a role for TNF could not be excluded, whereas in LPS pretreated animals TNF plays no part in sensitising to CX.

Expression of the calcium-binding protein S100A4 (p9Ka) in MMTV-neu transgenic mice induces metastasis of mammary tumours.

Increased levels of S100A4 (p9Ka) confer metastatic ability on a normally non-metastatic epithelial cell line. To find out whether S100A4 can induce metastasis in vivo, transgenic mice expressing high levels of S100A4, but which show no phenotypic effect, have been mated with MMTV-neu transgenic mice which succumb to stochastic mammary neoplasia related to expression of the MMTV-neu transgene. Resultant bitransgenic, multiparous, female progeny expressing both S100A4 and Neu have a slightly earlier incidence of palpable mammary tumours than the MMTV-neu offspring and specifically exhibit macroscopic metastatic lesions in the lungs. The S100A4 transgene is expressed in primary and secondary lesions of bitransgenic offspring and its expression is particularly associated with regions of invasion of primary lesions and metastases.

The role of enteric bacteria in the pathogenesis of fatal cycloheximide intolerance in mice pretreated with dexamethasone, promethazine or nordihydroguaiaretic acid.

As an incidental finding in a separate, ongoing investigation, dexamethasone was shown to sensitise mice fatally to a later challenge with a normally-tolerated dose of cycloheximide. This phenomenon is described here; it is also shown that two unrelated agents, namely promethazine and nordihydroguauaretic acid, duplicated this sensitising effect of the steroid. The three drugs have in common the ability to inhibit powerfully the synthesis of tumour necrosis factor-alpha in response to lipopolysaccharide, and it is suggested that this property is linked to their ability to induce fatal cycloheximide intolerance. Such drug-pretreated mice were protected if given dexamethasone at the time of cycloheximide challenge. An equal degree of protection was conferred on such animals by oral antibiotic treatment known to eliminate the aerobic intestinal flora. This indicated that the three agents induced fatal susceptibility to cycloheximide through the agency of the gut flora. It is proposed that the three drugs act by impairing the hepatic mechanism which normally removes portal vein-borne endogenous lipopolysaccharide, leading to systemic distribution of lipopolysaccharide, which is known from previous work, using a small dose of intraperitoneally injected lipopolysaccharide, to render mice fatally susceptible to a later cycloheximide challenge.

Immunocytochemical distribution of the calcium-binding protein p9Ka in normal rat tissues: variation in the cellular location in different tissues.

The family of S-100-related proteins consists of a number of small potential calcium-binding proteins of unknown function. Elevated expression of one of these proteins, p9Ka, or of its mRNA, correlates with the metastatic potential of cultured mammary epithelial cells from rat or mouse. Over-expression of p9Ka by transfection of benign rat mammary epithelial tumor cells with the gene for p9Ka induces the metastatic phenotype. At present there is little information on the occurrence of p9Ka in normal rat tissues. A specific antiserum immunocytochemically detects p9Ka intracellularly in most normal adult rat tissues studied, including smooth muscle, brown adipose tissue, and liver. In other tissues, p9Ka is localized specifically to some absorptive and keratinized epithelia, the acid-secreting parietal cells of the stomach, the neuronal cells within plexuses of the autonomic nervous system, and a proportion of cells of the immune system in spleen, lymph nodes, bone marrow, and blood. p9Ka is found widely in both arteries and veins, particularly in the smooth muscle and in the endothelium of smaller veins. In mammary gland, the pattern of staining suggests that p9Ka is extracellularly located in a region surrounding the ducts.

A prolonged period of uninterrupted protein synthesis is essential for survival in mice given a submicrogram dose of bacterial endotoxin.

A majority of mice failed to survive challenge with a normally tolerated dose of cycloheximide given at intervals up to 96 h after a single intraperitoneal injection of 0.02 microgram of S. enteritidis endotoxin. Only by 168 h did a majority of endotoxin-pretreated animals resist such a challenge. This finding underlines the essential role of uninterrupted protein synthesis in resistance to the lethal effects of endotoxin, and demonstrates the relatively persistent nature of this requirement after systemic exposure to an extremely low dose of lipopolysaccharide.

Cycloheximide or nordihydroguaiaretic acid protects mice against the lethal and hepatocytolytic effects of a combined challenge with D-galactosamine and bacterial endotoxin.

Both cycloheximide and nordihydroguaiaretic acid protect mice against the fatalities and associated panlobular hepatocyte necrosis that follow a challenge with D-galactosamine and bacterial endotoxin. It is proposed that cycloheximide acts as an inhibitor of the endotoxin-induced activation of phospholipase A2, thereby inhibiting the synthesis of leukotrienes which is now known to be a prerequisite for tumour necrosis factor-alpha (TNF) biosynthesis, the latter cytokine being regarded as the terminal mediator of the fatal D-galactosamine and endotoxin-induced syndrome. Nordihydroguaiaretic acid protection is explained by its inhibition of lipoxygenase enzymes, and thus ultimately of TNF production in response to endotoxin.

The effect of heparin on glomerular coagulation induced in mice by cycloheximide and bacterial lipopolysaccharide.

The glomerular capillary coagulation in mice which follows challenge with cycloheximide and a submicrogram dose of endotoxin was shown to become fully established between 4 h and 6 h after challenge. Complete protection against the renal lesion was achieved by heparin treatment 4 h after challenge. The antifibrinolytic agent, tranexamic acid, given 4 h and 6 h after challenge, abolished this heparin-induced protection, but had no effect when given 6 h and 8 h after challenge. These findings suggest that heparin in some way delayed the onset of cycloheximide and endotoxin-induced irreversible coagulation in glomerular capillaries, and by so doing, allowed the development of local, protective, fibrinolytic activity. A relatively brief period of such fibrinolytic activity appeared to confer lasting protection against coagulation in glomerular capillaries. Appropriately timed treatment with hydrocortisone also abolished the heparin-initiated protection, and the evidence suggests that the steroid acts by inhibiting the development of local fibrinolytic activity.

Hydrocortisone protects cycloheximide-challenged mice pretreated with Ehrlich ascites tumour cells or with cell-free Ehrlich ascites tumour fluid.

The invariably fatal outcome following cycloheximide challenge of mice pretreated 24 h earlier with either 1 x 10(7) washed Ehrlich ascites tumour cells i.v. or with 0.2 ml of cell-free Ehrlich ascites tumour fluid i.v. was prevented by hydrocortisone treatment. However, glucocorticosteroids failed to prevent the fatal outcome of cycloheximide challenge in mice bearing 5-day-old Ehrlich ascites tumours. The results are interpreted as indicating that there is a steroid demand which must be met to ensure survival of cycloheximide-challenged mice pretreated with tumour cells or with cell-free tumour ascites fluid and that an additional and essential requirement for uninterrupted protein synthesis exists in the case of mice with established ascites tumours and challenged with cycloheximide.

The effects of endotoxin pretreatment on subsequent challenge of mice with cycloheximide and a small dose of endotoxin.

Mice treated with 15.0 micrograms of S. enteritidis endotoxin are completely protected against the coagulopathic effects, but only partially protected against the fatal glucocorticoid deficiency which follow an otherwise lethal challenge with cycloheximide and 5.0 micrograms of the same endotoxin given 3 days later. Hydrocortisone treatment results in survival of all such tolerant, challenged animals. The protection conferred against occlusion of glomerular capillaries by fibrin coagula is abolished by EACA, suggesting that tolerance has induced high levels of fibrinolytic activity by the time of challenge, and evidence indicates that this protective degree of fibrinolytic activity persists for more than 24 h after cycloheximide and endotoxin challenge.

The roles of glucocorticosteroid and of fibrinolytic activity in the development of bilateral renal cortical necrosis in cycloheximide-treated mice challenged with a submicrogram dose of endotoxin.

Mice given cycloheximide and 0.2 microgram of endotoxin simultaneously developed ischaemic bilateral renal cortical necrosis as part of a fatal syndrome. Endotoxin given 2 h after cycloheximide, although fatal, failed to produce renal cortical necrosis. Investigation suggested that, following endotoxin challenge in cycloheximide-treated mice, the occurrence or non-occurrence of bilateral renal cortical necrosis was determined by the concentration of circulating glucocorticoids at the time of endotoxin challenge. Thus, below a certain, as yet undefined, glucocorticoid concentration, endotoxin does not cause renal cortical necrosis in cycloheximide-treated mice. Inhibition of the fibrinolytic system by epsilonaminocaproic acid (EACA) indicated that protection against renal cortical necrosis was directly mediated by increased fibrinolytic activity. Since this increased activity occurred during a period of profound cycloheximide-mediated inhibition of protein synthesis, it was postulated that pre-existing plasminogen activator was released from inhibition, and that this release could only occur when the glucocorticoid concentration fell below a certain critical value.

Further analysis of lethal factors in cycloheximide-treated mice given low doses of bacterial lipopolysaccharide.

Endotoxin challenge (0.2 micrograms per mouse) 6 h after a standard dose of cycloheximide was compared with the previously-reported effects of simultaneous injection of cycloheximide and endotoxin. With the submicrogram dose of endotoxin given 6 h after cycloheximide, fatalities occurred without evidence of thrombogenic bilateral renal cortical necrosis which characterized mice dying after the two agents were given together. Anticoagulation with heparin or with ancrod is life-saving, indicating that cycloheximide-treated mice were fatally susceptible to fibrinogen-to-fibrin conversion, and that, in contrast to the situation where cycloheximide and endotoxin are given simultaneously, there was no essential demand for supplementary glycocorticosteroid. A dose of 5.0 micrograms of endotoxin given 6 h after cycloheximide was fatal; again no renal cortical necrosis occurred, but both ancrod and hydrocortisone were essential to ensure survival.

Factors involved in the fatal susceptibility to submicrogram doses of endotoxin in cycloheximide-treated mice.

Analysis of fatal sensitivity to 0.2 microgram of S. enteritidis lipopolysaccharide in cycloheximide-treated mice identified two independent lethal elements. First, an absolute requirement for steroid supplementation to ensure survival suggests a crucial role for cycloheximide-mediated inhibition of steroidogenesis. The second factor is the development of virtually total bilateral renal cortical necrosis, itself a consequence of glomerular capillary occlusion with fibrin-like material. The survival of cycloheximide and endotoxin-challenged mice requires both hydrocortisone treatment and defibrination with ancrod. Cycloheximide and a smaller dose of endotoxin (0.1 microgram per mouse) is also fatal, but here steroid deficiency is not a crucial factor, protection being conferred by ancrod defibrination alone.

Fatal susceptibility to cycloheximide of mice bearing Ehrlich ascites tumours and of animals pretreated with cell-free Ehrlich ascites tumour fluid.

Soluble, but unidentified, factors in cell-free Ehrlich ascites tumour fluid induced, after a delay period, a fatal heparin- and ancrod-resistant susceptibility to a normally tolerated dose of cycloheximide. The evidence suggests that production of such factors is dependent upon interaction of tumour cells with the intravascular or the intraperitoneal compartment. The factors are considered likely also to play a crucial role in the similar heparin and ancrod-resistant fatalities which follow cycloheximide challenge of mice bearing established Ehrlich ascites tumours and in the deaths of mice challenged with cycloheximide 24 h after an intravenous injection of washed Ehrlich ascites tumour cells. These factors play no part in the heparin- and ancrod-preventable fatal syndrome following cycloheximide challenge either 2 h before or 2 h after intravenous injection of washed Ehrlich ascites tumour cells.

Uninterrupted protein synthesis is essential for survival in the early stages of carbon tetrachloride-induced hepatocellular necrosis in the mouse.

The fatal syndrome produced by cycloheximide given 6 h after a hepatonecrogenic dose of CCl4 is due neither to direct toxic synergism between CCl4 and cycloheximide nor to transient sinusoidal thrombosis. It is suggested that survival in the presence of unknown factors released from dying liver cells requires uninterrupted protein synthesis. The life-saving effect of sterilization of the intestine by antibiotics indicates that the gut flora or its products play a vital role in pathogenesis.

The effect of cycloheximide, heparin and ancrod on mice given Ehrlich ascites tumour cells intravenously.

Mice injected intra-venously with Ehrlich ascites tumour cells showed a fatal susceptibility to normally-tolerated doses of cycloheximide when the latter was given at the following times after the tumour cells--2 h, 24 h, 48 h, and 72 h. When the interval between tumour cell injection and cycloheximide challenge was extended to 1 week or 3 weeks (at which latter time mice have clinical "metastatic" neoplasia), no such susceptibility was shown. In the case of the 2 h interval between tumour cells and cycloheximide (and also when cycloheximide was given 2 h before tumour cells), the lethal syndrome was prevented by heparin treatment and also by Ancrod defibrination. This strongly suggests that heparin is effective by virtue of its anticoagulant properties, and that fibrinogen-to-fibrin conversion is an essential step in the pathogenesis. Neither heparin nor Ancrod confer any protection when used in the syndrome consequent upon cycloheximide challenge 24 h after tumour cell injection, which indicates that, in this case, the conversion of fibrinogen to fibrin is not an essential step in determining the fatal outcome.

A lethal syndrome in mice following administration of carbon tetrachloride and cycloheximide, and its prevention by heparin treatment.

When a hepatotoxic dose of CCl4 is followed in 6 h (but not in 18 h) by 30 micrograms per g body weight of cycloheximide, a lethal, shock-like state develops. This is prevented by heparin treatment. This lethal syndrome is compared with other, similar, induced lethal states in which cycloheximide plays an essential role, and in which heparin is lifesaving. It is postulated that, after CCl4, a phase of procoagulant activity occurs in the dying centrilobular zone hepatocytes, but that unimpaired protein synthesis permits responsive release of endogenous heparin and thereby prevents thrombosis in centrilobular sinusoids. Cycloheximide is thought to inhibit this heparin release and to allow a transient episode of occlusive centrilobular microthrombosis with consequent irreversible ischaemic damage to the mid-gut.

Cycloheximide challenge of mice bearing subcutaneous tumours, with observations on heparin treatment.

Mice bearing large subcutaneous transplants of ovarian teratocarcinoma succumb when given large doses of cycloheximide, whereas animals with equally large subcutaneous Ehrlich tumours are resistant to this treatment. Adequate dosage of heparin is life-saving in cycloheximide-treated, teratoma-bearing mice. The results are discussed in the context of previous work, and one tentative suggestion made is that cycloheximide interferes with the mechanism for release of endogenous heparin which is an essential component of the host response to a growing teratocarcinoma.

The lethal syndrome following intravenous injection of Ehrlich ascites tumour cells in cycloheximide-treated mice and its prevention by heparin.

Heparin was shown to be a life-saving treatment in the otherwise lethal syndrome following intravenous injection of Ehrlich ascites tumour cells in cycloheximide-treated mice, and in mice treated with cycloheximide 2 h after injection of Ehrlich tumour cells. The effectiveness of heparin cannot be attributed to interference with initial arrest of tumour cells. A similar lethal syndrome, again prevented by heparin treatment, occurs in mice given intravenous colloidal carbon and cycloheximide. It is suggested that large doses of intravenous particulates bring about changes in plasma composition which are rapidly rectified in an otherwise normal mouse, but which persist under the influence of cycloheximide and lead to the irreversible and fatal outcome. Finally, evidence is presented which suggests that liver protein synthesis may play an essential role in the period following intravenous injection of tumour cells in an otherwise normal mouse.