Hypoxia, RONS and energy metabolism in articular cartilage.

OBJECTIVE: Increased pro-inflammatory cytokines and reactive oxygen and nitrogen species (RONS) occur in osteoarthritis (OA). Oxygen tension can alter the levels of RONS induced by interleukin-1 (IL-1). RONS such as nitric oxide (NO) can alter energy metabolism. The aim of this study was to determine if oxygen tension alters energy metabolism, in articular cartilage, in response to IL-1 or NO and to determine if cell death occurred. DESIGN: Porcine articular chondrocytes were incubated with IL-1 or the NO donor NOC-18 for 48 h in either 1, 5 or 20% O(2). Adenosine triphosphate (ATP) levels were measured and immunoblots for adenosine monophosphate-activated protein kinase (AMPK) were done. Protein translation was measured by S6 activation. Senescence and autophagy were determined by increased caveolin or conversion of LC3-I to LC3-II respectively. RESULTS: One percent O(2) significantly reduced ATP levels compared with 20% O(2). Five percent O(2) significantly increased ATP levels compared with 20% O(2). One percent O(2) significantly increased phospho-AMPK (pAMPK) protein expression compared with 5 or 20% O(2). Oxygen tension had no effects on pS6, caveolin or LC3-II levels. IL-1-induced NO production was significantly reduced with decreased oxygen tension, and significantly reduced ATP levels at all oxygen tensions, but pAMPK was only significantly increased at 5% O(2). IL-1 significantly reduced pS6 at all oxygen tensions. IL-1 had no effects on caveolin and significantly increased LC3-II at 20% O(2) only. NOC-18 significantly reduced ATP levels at all oxygen tensions, and significantly increased pAMPK at 5% O(2) only, and significantly decreased pAMPK at 1% O(2). NOC-18 significantly reduced pS6 at 1% O(2) and significantly increased caveolin at 5% O(2), and LC3-II at 1% O(2). CONCLUSION: Our data suggest 5% O(2) is optimal for energy metabolism and protective to some effects of IL-1 and NO. NO has the greatest effects on ATP levels and the induction of autophagy at 1% O(2).

Reactive nitrogen and oxygen species in interleukin-1-mediated DNA damage associated with osteoarthritis.

OBJECTIVE: Osteoarthritis (OA) is associated with increased levels of reactive nitrogen and oxygen species and pro-inflammatory cytokines, such as interleukin-1 (IL-1). Nitric oxide (NO) can mediate a number of the catabolic effects of IL-1 in articular cartilage. The aims of this study were to determine if OA cartilage shows evidence of DNA damage, and if IL-1 could induce DNA damage in non-OA cartilage by increasing NO or superoxide. METHODS: Articular chondrocytes were isolated from porcine femoral condyles and embedded in 1.2% alginate. The effects of 24h incubation with IL-1, the nitric oxide synthase 2 (NOS2)-selective inhibitor, the free radical scavenger superoxide dismutase (SOD), the NO donor NOC18, or the combined NO and peroxynitrite donor SIN-1 on DNA damage were tested, using the "comet" assay. NO production was measured using the Griess assay. The type of oxidative damage present was assessed using a modified comet assay. RESULTS: OA cartilage had significantly more DNA damage than non-OA cartilage (P<0.001). IL-1 caused an increase in DNA damage (P<0.01), which was associated with increased NO production (P<0.01). Both oxidative DNA strand breaks and base modifications of purines and pyrimidines were observed. IL-1-induced DNA damage was inhibited by an NOS2 inhibitor or by SOD (P<0.01). Furthermore, NOC18 or SIN-1 caused DNA damage (P<0.001). CONCLUSION: Our work shows chondrocytes in osteoarthritic cartilage exhibit DNA damage, and that IL-1 induces DNA damage and reactive oxygen and nitrogen species in non-OA chondrocytes in alginate.

Oxygen, nitric oxide and articular cartilage.

Molecular oxygen is required for the production of nitric oxide (NO), a pro-inflammatory mediator that is associated with osteoarthritis and rheumatoid arthritis. To date there has been little consideration of the role of oxygen tension in the regulation of nitric oxide production associated with arthritis. Oxygen tension may be particularly relevant to articular cartilage since it is avascular and therefore exists at a reduced oxygen tension. The superficial zone exists at approximately 6% O2, while the deep zone exists at less than 1% O2. Furthermore, oxygen tension can alter matrix synthesis, and the material properties of articular cartilage in vitro. The increase in nitric oxide associated with arthritis can be caused by pro-inflammatory cytokines and mechanical stress. Oxygen tension significantly alters endogenous NO production in articular cartilage, as well as the stimulation of NO in response to both mechanical loading and pro-inflammatory cytokines. Mechanical loading and pro-inflammatory cytokines also increase the production of prostaglandin E2 (PGE2). There is a complex interaction between NO and PGE2, and oxygen tension can alter this interaction. These findings suggest that the relatively low levels of oxygen within the joint may have significant influences on the metabolic activity, and inflammatory response of cartilage as compared to ambient levels. A better understanding of the role of oxygen in the production of inflammatory mediators in response to mechanical loading, or pro-inflammatory cytokines, may aid in the development of strategies for therapeutic intervention in arthritis.

The influence of mechanical compression on the induction of osteoarthritis-related biomarkers in articular cartilage explants.

OBJECTIVE: Macromolecules of the articular cartilage extracellular matrix released into synovial fluid, blood, or urine can serve as potentially useful biomarkers of the severity of osteoarthritis (OA). Biomechanical factors play an important role in OA pathogenesis, yet their influence on biomarker production is not well understood. The goal of this study was to examine the hypothesis that dynamic mechanical stress influences the release of these biomarkers from articular cartilage. METHODS: Explants of porcine cartilage were subjected to dynamic compression at 0.5 Hz for 24h at stresses ranging from 0.006 to 0.1 MPa. The concentrations of cartilage oligomeric matrix protein (COMP), keratan sulfate (KS measured as the 5 D 4 epitope), total sulfated glycosaminoglycan (S-GAG), and the KS (keratanase-digestible) and chondroitin sulfate (CS) (chondroitinase-digestible) fractions of S-GAG were measured. Radiolabel incorporation was used to determine the rates of proteoglycan and protein synthesis. RESULTS: The magnitudes of mechanical stress applied in this study induced nominal tissue strains of 4-23%, consistent with a range of physiological to hyperphysiologic strains measured in situ. COMP release increased in proportion to the magnitude of dynamic mechanical stress, while KS, CS and total S-GAG release increased in a bimodal pattern with increasing stress. Protein and proteoglycan synthesis were significantly decreased at the highest level of stress. CONCLUSION: Mechanical stress differentially regulates the turnover of distinct pools of cartilage macromolecules. These findings indicate that mechanical factors, independent of exogenous cytokines or other stimulatory factors, can influence the production and release of OA-related biomarkers from articular cartilage.

Induction of cyclooxygenase-2 by mechanical stress through a nitric oxide-regulated pathway.

OBJECTIVE: Biomechanical signals play important roles in regulating the homeostasis of articular cartilage, but under abnormal conditions may be a critical factor in the onset and progression of arthritis. Prostaglandin E(2) (PGE(2)) and nitric oxide (NO), derived from the enzymes cyclo-oxygenase 2 (COX2) and NO synthase 2 (NOS2), are inflammatory mediators that modulate numerous physiological and pathophysiological processes and are potentially important pharmacological targets in osteoarthritis. The goal of this study was to determine the effect of mechanical compression on PGE(2) production in the presence of selective NOS2 and COX2 inhibitors. METHODS: Articular cartilage explants harvested from 2-3-year-old pigs were subjected to intermittent compression at 0.5Hz over a range of stress magnitudes. PGE(2) and NO production into the media were determined in the presence and absence of the NOS2 inhibitor 1400W or the COX2 inhibitor NS398. COX2 protein levels were determined by immunoblot analysis. RESULTS: Mechanical compression significantly increased NO and PGE(2) synthesis in a manner that was dependent on the magnitude of stress. The selective COX2 inhibitor blocked compression-induced NO and PGE(2) production. Compression in the presence of 1400W further increased COX2 expression resulting in a 10-fold increase in PGE(2) production compared to uncompressed explants with 1400W and a 40-fold increase in PGE(2) compared to uncompressed explants without 1400W. CONCLUSION: Mechanical compression of articular cartilage increased COX2 and PGE(2) production through a NO-dependent pathway, and therefore pharmacological agents that target the NOS2 pathway in cartilage may have a significant influence on prostanoid production in the joint.

Interleukin-1, tumor necrosis factor alpha, and interleukin-17 synergistically up-regulate nitric oxide and prostaglandin E2 production in explants of human osteoarthritic knee menisci.

OBJECTIVE: In osteoarthritis (OA), a combination of biochemical and biomechanical factors may damage both menisci and articular cartilage. Nitric oxide (NO) and prostaglandin E2 (PGE2) have been implicated as mediators of inflammation in OA. The goals of this study were to determine if menisci from patients with OA produce NO and PGE2, and if the proinflammatory cytokines interleukin-1beta (IL-1beta), tumor necrosis factor a (TNFalpha), and IL-17 augment NO and PGE2 production by these tissues. METHODS: Menisci were obtained from 17 patients (age 47-75 years) undergoing total knee replacement for OA. Tissue explants were cultured alone or with IL-1beta, IL-17, or TNFalpha, and the release of NO and PGE2 from the tissue as well as the presence of type 2 nitric oxide synthase (NOS2) and cyclooxygenase 2 (COX-2) antigens were measured. RESULTS: All menisci constitutively produced NO, and significant increases in NO production were observed in the presence of IL-1beta, TNFalpha, or IL-17 (P < 0.05). The combination of IL-17 and TNFalpha significantly increased NO production compared with either cytokine alone. Basal and cytokine-stimulated NO synthesis was inhibited by the NOS inhibitors NG-monomethyl-L-arginine or N-3-aminoethylbenzylacetamidine (1400W). IL-1beta significantly increased PGE2 production. The combination of IL-1beta and TNFalpha had an additive effect on PGE2 production, while addition of IL-17 to TNFalpha or IL-1beta synergistically enhanced PGE2 production. Inhibition of NO production by 1400W significantly increased IL-1beta-stimulated PGE2 production, and inhibition of PGE2 production by the COX-2 inhibitor N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide significantly increased IL-17-stimulated NO production. CONCLUSION: Menisci from humans with OA spontaneously produced NO and PGE2 in a manner that was synergistically or additively augmented by cytokines. NO and PGE2 exhibited reciprocal regulatory effects on one another, suggesting that pharmaceutical agents designed to inhibit NOS2 or COX-2 production may in fact be influencing both pathways.

The effects of static and intermittent compression on nitric oxide production in articular cartilage explants.

Nitric oxide (NO) production and NO synthase (NOS) expression are increased in osteoarthritis and rheumatoid arthritis, suggesting that NO may play a role in the destruction of articular cartilage. To test the hypothesis that mechanical stress may increase NO production by chondrocytes, we measured the effects of physiological levels of static and intermittent compression on NOS activity, NO production, and NOS antigen expression by porcine articular cartilage explants. Static compression significantly increased NO production at 0.1 MPa stress for 24 h (P < 0.05). Intermittent compression at 0.5 Hz for 6 h followed by 18 h recovery also increased NO production and NOS activity at 1.0 MPa stress (P < 0.05). Intermittent compression at 0.5 Hz for 24 h at a magnitude of 0.1 or 0.5 MPa caused an increase in NO production and NOS activity (P < 0.05). Immunoblot analysis showed stress-induced upregulation of NOS2, but not NOS1 or NOS3. There was no loss in cell viability following any of the loading regimens. Addition of 2 mM 1400 W (a specific NOS2 inhibitor) reduced NO production by 51% with no loss of cell viability. These findings indicate that NO production by chondrocytes is influenced by mechanical compression in vitro and suggest that biomechanical factors may in part regulate NO production in vivo.

Mechanical stress and nitric oxide influence leukotriene production in cartilage.

Nitric oxide (NO) and leukotrienes regulate a variety of processes in joint tissues and are frequently elevated in arthritis. Mechanical stress can induce biochemical and functional changes in cartilage that may influence mediator production. To investigate the relationship between mechanical stress and the production of leukotriene B(4) (LTB(4)) and NO, explants of porcine articular cartilage were subjected to mechanical compression for 1 h followed by 23 h recovery in the presence or absence of the NOS2 inhibitor 1400W. Dynamic compression significantly increased LTB(4) and LOX protein production in the presence of 1400W. The induced LTB(4) was functional as evidenced by its ability to promote chemotaxis of RBL-2H3 cells expressing the LTB(4) receptor. Increased LOX protein but not LTB(4) occurred in response to compression alone. These findings provide a direct link between mechanical stress and inflammation in cartilage and may have implications in the pathogenesis and treatment of arthritis.

The effect of dynamic mechanical compression on nitric oxide production in the meniscus.

OBJECTIVE: The menisci play an important role in the biomechanics of the knee, and loss of meniscal function has been associated with progressive degenerative changes of the joint in rheumatoid arthritis as well as in osteoarthritis. However, little is known about the underlying mechanisms that link meniscal injury or degeneration to arthritis. Meniscal fibrochondrocytes respond to environmental mediators such as growth factors and cytokines, but the influence of mechanical stress on their metabolic activity is not well understood. Nitric oxide (NO) is believed to play a role in mechanical signal transduction, and there is also significant evidence of its role in cartilage and meniscus degeneration. The goal of this study was to determine if meniscal fibrochondrocytes respond to mechanical stress by increasing NO production in vitro. DESIGN: Explants of lateral and medial porcine menisci were dynamically compressed in a precisely controlled manner, and NO production, nitric oxide synthase antigen expression and cell viability were measured. The relative responses of the meniscal surface and deep layers to dynamic compression were also investigated separately. RESULTS: Meniscal NO production was significantly (P< 0.01) increased by dynamic compression in both the medial and lateral menisci. Dynamically compressed menisci contained inducible nitric oxide synthase antigen, while uncompressed menisci did not. Significant (P< 0.05) zonal differences were observed in basal and compression-induced NO production. DISCUSSION: Our findings provide direct evidence that dynamic mechanical stress influences the biological activity of meniscal cells. These results suggest that NO production in vivo may be in part regulated by mechanical stress acting upon the menisci. Since NO affects matrix metabolism in various intraarticular tissues, alterations in the distribution and magnitude of stress in the menisci may have important metabolic as well as biomechanical consequences on joint physiology and function.

Primary human osteoblast proliferation and prostaglandin E2 release in response to mechanical strain in vitro.

The application of mechanical loads to bone cells in vitro has been found to generate variable responses, which may in part be due to the source of the cell used and the characteristics of the strain applied. The aim of this study was to establish a system for applying well-defined physiological levels of mechanical strain to a well-defined population of human osteoblast-like cells. Human bone-derived cells obtained from the greater trochanter of the femur during total hip arthroplasty for osteoarthritis were cultured in the presence of 10 nmol/L dexamethasone and 100 mumol/L L-ascorbate-2-phosphate. Replicates of cells from each patient were loaded on separate occasions using controlled cyclical strains of 4000 microstrain (mu epsilon) or less. Strain gauges recorded reliable, reproducible strains between 1000 and 6000 mu epsilon. To establish reproducibility, sequential explant cultures derived from two patients were studied. A consistent increase (p < 0.05) in proliferation between replicates and explants derived from one patient subjected to 1600 mu epsilon on separate occasions was observed. Cells derived from sequential explants of the second patient showed no consistent increase in proliferation between replicates and explants. Three of six patients showed a significant increase (p < 0.05) in PGE2 production after 5 h in response to stretch (4000 mu epsilon) in all replicates on separate occasions, whereas, in the other three populations of cells, no increase in PGE2 was measured in any of the replicates. These results show that the application of highly controlled strains causes a significant effect on human bone cells, but only in a proportion of subjects. The response is consistent between sequential explants derived from the same patient. The implications of this study are that human osteoblast-like cells do respond to physiological strain in vitro, although some cells are more strain sensitive than others.

Expression of cation exchanger NHE and anion exchanger AE isoforms in primary human bone-derived osteoblasts.

The authors used isoform-specific antibodies against cation (NHE) and anion (AE) exchange isoforms in order to establish their specific expression and localization in dispersed human bone-derived cells. Immunocytochemical preparations of permeabilized osteoblasts probed with polyclonal antibodies were optically analysed by conventional immunofluorescence and con-focal laser scanning microscopy. These techniques demonstrated the abundant presence of epitopes of the cation exchangers NHE1 and NHE3 and the anion exchanger AE2 in these cells. The NHE1 and NHE3 isoform proteins were predominantly located in subplasmalemmal and nucleoplasmic vesicles. The AE2 isoform was densely localized to a subcellular location characteristic of the Golgi complex. The molecular identity of the AE and NHE isoforms was investigated by RT-PCR that confirmed the presence of NHE1 and NHE3 transcripts in addition to NHE4. RT-PCR and diagnostic restriction analysis of amplified AE cDNA established preferential AE2 expression. Since AE2 has been shown to act as a sulfate transporter at low pH, it is possible that it performs this function in the osteoblast Golgi complex where sulfation reactions occur post-translationally on numerous extracellular matrix macromolecules prior to secretion and mineralization. The Na(+)/H(+)exchanger proteins are regulated by mitogenic and non-mitogenic stimuli in the osseus environment and are involved in the large fluxes of ions and protons that necessarily occur during bone formation and resorption and thus play an important role in intracellular ion homeostasis in osteoblasts.

Proliferation and collagen synthesis of human anterior cruciate ligament cells in vitro: effects of ascorbate-2-phosphate, dexamethasone and oxygen tension.

Clinical and experimental studies demonstrate that injured anterior cruciate ligaments (ACL) do not usually heal and that autografts used to repair the ACL rapidly weaken in the early period and take a long time to regain strength. The aim of this study was to develop an in vitro culture system in which environmental and biochemical factors influencing the proliferation and matrix synthesis of cells derived from human anterior cruciate ligaments can be studied. Primary cultures of human ACL cells were obtained by outgrowth from explants of normal ACL obtained at knee replacement for osteoarthritis in Dulbecco's minimum essential medium (DMEM). The effects of the additives 100 microm L-ascorbic acid-2-phosphate (Asc-2-P) and 10 n m dexamethasone (dex) on proliferation and collagen synthesis were assessed after 4, 8 and 12 days in culture. Ligament cells were grown at 0, 5, 10 and 21%p O(2)in the presence of 100 microm asc-2-P and 10 n m dex. DNA content was assessed using the Hoechst dye method and collagen synthesis by the incorporation of 5 mCi/ml [(3)H]proline after 3, 6 and 12 days in culture. At 21%p O(2), the presence of asc-2-P and dex induced significantly greater (P< 0.01, ANOVA) cell proliferation than with either additives alone. Greatest percentage collagen to total protein synthesis was observed when cells were grown in the presence of asc-2-P only. Least proliferation and percentage collagen to total protein synthesis was seen when both additives were omitted. Greatest cell proliferation was seen when cells were grown in 10%p O(2)and 5%p O(2)was associated with increased collagen synthesis. These results suggest that it is possible to study the effects of environmental and biochemical factors on human ACL healing in vitro. Our data suggest oxygen can influence certain biosynthetic activities of ACL cells. Low oxygen tensions lead to an increase in collagen production by ACL cells. However early responses to injury require extensive cell proliferation which may be activated at higher p O(2). Variation of p O(2)in ligaments during healing may therefore be an important modulator of successful repair.

PTH/PTHrP receptor expression on osteoblasts and osteocytes but not resorbing bone surfaces in growing rats.

Using in situ hybridization, we correlated the expression of mRNA for the parathyroid hormone/parathyroid hormone related peptide (PTH/PTHrP) receptor with bone formation and resorption in undecalcified serial sections of bones from growing rats. In addition we investigated the presence of biologically active receptors in the same locations using an in vivo autoradiographic technique. In the ulnae of growing rats, there are well defined zones of cortical bone formation and resorption. These contribute to the modeling drifts by which the bone achieves its adult shape. Forming surfaces incorporate fluorochrome labels, are lined with osteoid, and have a layer of cuboidal osteoblasts that have a high alkaline phosphatase activity. Resorbing surfaces have no fluorochrome incorporation, no osteoid, and are lined with resorbing cells with high tartrate-resistant acid phosphatase (TRAP) activity. PTH/PTHrP receptor mRNA was expressed predominantly on forming but not on resorbing bone surfaces and colocalized with sites of binding of radiolabeled PTH after intravenous injection. PTH/PTHrP mRNA expression on osteocytes was inconclusive but radiolabeled PTH bound to a proportion of osteocytes in all regions of the cortex although binding was not specifically related to areas of bone formation or resorption. These results suggest that in growing animals the actions of PTH or PTHrP are connected more with bone formation than resorption. Such a role may be linked to the ability of PTH to induce bone formation in adults but does not explain the actions of the hormone in regulating resorption. Binding of PTH to osteocytes increases the evidence for a physiological role for these cells.

Partial characterization of a cDNA for human stearoyl-CoA desaturase and changes in its mRNA expression in some normal and malignant tissues.

A segment of 712 bases coding for part of the human stearoyl-CoA desaturase gene was made by polymerase chain reaction (PCR) using primers based on published rat cDNA sequences. The human PCR product was confirmed by DNA sequencing. It was next cloned into a vector from which anti-sense, highly radioactive RNA transcripts were made in vitro using T7 polymerase. The transcripts were used to probe desaturase mRNA in a number of human tumour and control tissues, using a very sensitive solution hybridization/RNase protection assay. Increased desaturase mRNA levels were found in colonic and oesophageal carcinomas and in hepatocellular adenoma; however, no consistent trend was seen in hepatocellular carcinoma. It is suggested that certain classes of tumour may exhibit increased levels of desaturase mRNA.

Fatty acid composition of normal and malignant cells and cytotoxicity of stearic, oleic and sterculic acids in vitro.

The aim of this study was to investigate the hypothesis that saturated fatty acids are differentially cytotoxic to cancer cells. Three studies were undertaken to: (1) measure the toxicities of stearic and oleic acids to normal and malignant cells in vitro, (2) assess if there is any relationship between toxicity and relative fatty acid composition and (3) determine whether the relative fatty acid composition of a cancer cell line could be modified by sterculic acid, an inhibitor of delta-9-desaturase. Stearic (18:0) and oleic (18:1) acids inhibited the colony-forming abilities of five human cancer cell lines and two non-neoplastic cell lines in a dose-dependent fashion. The concentration of oleic acid required to reduce colony formation ability by 50% was 2.5-6.0-fold greater than that of stearic acid. Addition of sterculic acid to a cancer cell line resulted in steady-state levels of stearic acid and increasing percentage of oleic acid.

Manipulation of body fat composition with sterculic acid can inhibit mammary carcinomas in vivo.

Sterculic acid, a delta-9-desaturase inhibitor, administered to rats caused a rise in the stearic:oleic acid ratio of total lipids in peripheral red cells, serum and liver (P less than 0.001). As a reduction in the stearic:oleic acid ratio has been described in cancer cells, we investigated the effect of sterculic acid on tumour growth. Female F344 rats were injected subcutaneously with two different doses of sterculic acid for 4 weeks prior to, and 4 weeks following, implantation of a nitrosomethylurea-induced mammary tumour. Tumour growth was inhibited equally by the two doses of sterculic acid (P less than 0.001). A rise in the stearic:oleic acid ratio of tumours was observed in rats treated for only 16 days with sterculic acid. Manipulation of the tissue stearic:oleic acid ratio inhibits transplanted mammary tumour growth in rats.

Evaluation of the therapeutic use of radioactive monoclonal antibodies C242 and C215 in transplanted murine tumours.

The therapeutic roles of monoclonal antibodies C242 and C215 labelled with 131I were investigated in transplanted mammary carcinomas. Fragments (10 mg) of mammary tumour from (A x Pc) F1 hybrid mice were implanted subcutaneously in 72 mice of the same strain. Twelve mice were injected with 131I-C242 starting from day 12 following tumour implantation, and eight out of 12 survived for 60 days. By contrast all but one of 36 control mice died within the same time period (P less than 0.0001). Twelve mice were injected with 131I-C215. Seven out of the 12 were alive at 60 days whilst all but 1 of 36 control animals had died (P less than 0.0001). In this study targeted immunotherapy using 131I-labelled antibodies C242 and C215 inhibited tumour growth and prolonged survival. Measurements of tumour area showed a 27-39 percent reduction in the mice receiving the radiolabelled conjugates C242 and C215 (P less than 0.004 and P = 0.028 respectively).

Stearic acid and carcinogenesis.

Decreased membrane rigidity is one of the characteristics of malignant cells, resulting in part from the desaturation of stearic acid into oleic acid. In this study we investigated the influence of stearic acid on tumour cell inhibition in vitro and tumour development in vivo. Stearic acid inhibited the colony-forming ability of 4 out of 5 rat and two human tumour continuous cell lines in vitro. In contrast, the colony-forming ability of rat fibroblasts was not inhibited and that of human foetal lung fibroblasts was inhibited at a higher dose than that required to inhibit human tumour cell lines. Using a model of rat mammary carcinoma induced by nitroso-methyl urea (NMU) the subcutaneous injection of stearic acid at weekly intervals prevented tumour development in 5 to 10 rats. Using iodostearic acid twice weekly, 11 of 19 rats were alive and tumour free at week 22 whilst all of 14 animals injected with NMU alone had died of tumour by the 16th week. The ratio of stearic to oleic acids in erythrocyte membranes was significantly reduced in the tumour-bearing rats, but was normal in tumour-free animals treated with stearic or iodostearic acid. These preliminary data indicate that stearic acid inhibits tumour development in rats.

Proliferative instability and experimental carcinogenesis at colonic anastomoses.

The possibility that proliferative instability around a healing anastomosis promotes carcinogenesis was tested in 234 male Sprague-Dawley rats. Animals received the first of five weekly injections of azoxymethane (total dose 50 mg/kg) either immediately after transection of the descending colon or at 2, 4, 8, and 12 weeks later; controls received handling of the bowel alone. Crypt cell proliferation was assessed by autoradiography following 3HTdR injection. An overall increase in tumour yields in all transection groups was due solely to the frequent presence of anastomotic tumours. Changes in crypt morphometry and labelling index were most marked in crypt positions 1-10 away from the anastomosis. Crypts at this site increased in height at 2, 4, and 8 weeks (p less than 0.001) but returned to normal values by 12 weeks. Likewise, labelling index was increased at 2, 4, and 8 weeks (p less than 0.001) and remained higher at 12 weeks (p less than 0.05). Increased crypt cell proliferation in the immediate vicinity of an apparently 'healed' colonic anastomosis may explain its persisting susceptibility to carcinogenesis.