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Background: A careful analysis of the reasons for ACL reconstruction failure is essential to selection of the optimal surgical revision technique designed to ensure good rotational stability and to minimise the risk of re-rupture., Objective: To evaluate anterolateral ligament (ALL) stabilisation during revision ACL reconstruction., Hypothesis: ALL stabilisation during revision ACL reconstruction provides good rotational stability without increasing the risk of complications., Material and Methods: This multicentre study included 349 patients, 151 retrospectively and 198 prospectively. There were 283 males and 66 females. Inclusion criteria were an indication for revision ACL reconstruction surgery with combined intra-articular reconstruction and ALL stabilisation after failed autograft ACL reconstruction, and intact PCL. Exclusion criteria were primary ACL reconstruction and concomitant peripheral medial and/or lateral lesions. Each patient underwent a clinical and radiographic evaluation before and after revision surgery. Before revision surgery, the mean IKDC score was 56.5±15.5 and 96% of patients were IKDC C or D., Results: Rates were 5.0% for early and 10.5% for late postoperative complications. Lachmann's test had a hard stop at last follow-up in 97% of patients. The pivot-shift test was positive in 1% of patients. The mean subjective IKDC score was 84.5±13.0 and 86.5% of patients were IKDC A or B. The proportions of patients with radiographic knee osteoarthritis at last follow-up was unchanged for the lateral tibio-femoral and patello-femoral compartments but increased by 9.7% to 21.2% for the medial tibio-femoral compartment. The re-rupture rate was 1.2% and the further surgical revision rate was 5.4%., Conclusion: Anterior laxity at last follow-up was consistent with previous studies of revision ACL reconstruction. However, rotational stability and the re-rupture risk were improved. ALL stabilisation is among the techniques that deserve consideration as part of the therapeutic options for revision ACL reconstruction., Level of Evidence: IV, retrospective and prospective cohort study., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Introduction: Lateral tenodesis (LT) is performed to limit the risk of iterative tear following anterior cruciate ligament (ACL) reconstruction in at-risk patients. By adding an extra procedure to isolated ACL graft, LT reconstruction increases operating time and may complicate postoperative course. The objective of the present study was to evaluate the rate of early complications. The study hypothesis was that associating ALL reconstruction to ACL reconstruction does not increase the complications rate found with isolated ACL reconstruction., Material and Methods: A prospective multicenter study included 392 patients: 70% male; mean age, 29.9 years; treated by associated ACL and LT reconstruction. All adverse events were inventoried., Results: Mean hospital stay was 2 days, with 46% day-surgery. Walking was resumed at a mean 27 days, with an advantage for patients treated by the hamstring technique. The early postoperative complications rate was 12%, with 1.7% specifically implicating LT reconstruction: pain, hematoma, stiffness in flexion and extension, and infection. There was a 5% rate of surgical revision during the first year, predominantly comprising arthrolysis for extension deficit. The 1-year recurrence rate was 2.8%., Discussion: The complications rate for combined intra- and extra-articular reconstruction was no higher than for isolated intra-articular ACL reconstruction, with no increase in infection or stiffness rates. The rate of complications specific to ALL reconstruction was low, at 1.7%, and mainly involved fixation error causing lateral soft-tissue impingement., Level of Evidence: IV, prospective multicenter study., (Copyright © 2017. Published by Elsevier Masson SAS.)

Background: Patient-specific cutting guides were recently introduced to facilitate total knee arthroplasty (TKA). Their accuracy in achieving optimal implant alignment remains controversial. The objective of this study was to evaluate postoperative radiographic outcomes of 50 TKA procedures with special attention to posterior tibial slope (PTS), which is difficult to control intraoperatively. We hypothesized that patient-specific cutting guides failed to consistently produce the planned PTS., Material and Methods: The Signature™ patient-specific cutting guides (Biomet) developed from magnetic resonance imaging data were used in a prospective case-series of 50 TKAs. The target PTS was 2°. Standardised digitised radiographs were obtained postoperatively and evaluated by an independent reader. Reproducibility of the radiographic measurements was assessed on 20 cases. The posterior cortical line of the proximal tibia was chosen as the reference for PTS measurement. Inaccuracy was defined as an at least 2° difference in either direction compared to the target., Results: The implant PTS was within 2° of the target in 72% of knees. In the remaining 28%, PTS was either excessive (n=10; maximum, 9°) or reversed (n=4; maximum, -6°). The postoperative hip-knee-ankle angle was 0° ± 3° in 88% of knees, and the greatest deviation was 9° of varus., Conclusion: These findings support our hypothesis that patient-specific instrumentation decreases PTS accuracy. They are consistent with recently published data. In contrast, patient-specific instrumentation provided accurate alignment in the coronal plane., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

Purpose: The purpose of this study was to test the hypothesis that the surgical technique using alternating fluid irrigation and carbon dioxide (CO2) gas medium as a means of knee joint distension during arthroscopy is a safe and effective method, compared to traditional fluid arthroscopy., Methods: This prospective randomized comparative study involved 94 patients undergoing the same arthroscopic ACL reconstruction surgical procedure except for the medium used to distend the joint: In a first group of 48 patients ("ACL gas" group), CO2 insufflation was mainly used, at a low pressure of 40-50 mmHg, alternating with a fluid medium for knee joint washout only. In the second group of 46 patients, classical arthroscopic joint distension by fluid was used, with a pump pressure of 50-70 mmHg. Early pre- and post-operative complications, duration of surgery, intraoperative monitoring data and particularly the end-tidal carbon dioxide (EtCO2) as a marker of CO2 blood diffusion were prospectively collected., Results: EtCO2 and other monitoring data changes before and after tourniquet release were not different between the two groups. Tourniquet time was significantly longer when using fluid rather than gas. We observed 3 cases of small and localized subcutaneous emphysema, resolving completely within hours. Hematoma and reflex sympathetic dystrophy occurred more often in the "ACL fluid" group, with no statistical significance., Conclusion: Low-pressure CO2 knee joint insufflation proved to be a safe technique capable of improving surgical comfort compared with the use of fluid alone. The CO2 blood diffusion measured by end-tidal carbon dioxide monitoring during ACL arthroscopic reconstruction was similar with or without gas insufflation.

Background: For over a decade, computer-assisted orthopaedic surgery for total knee arthroplasty has been accepted as ensuring accurate implant alignment in the coronal plane., Hypothesis: We hypothesised that lack of accuracy in skeletal landmark identification during the acquisition phase and/or measurement variability of the infrared optical system may limit the validity of the numerical information used to guide the surgical procedure., Methods: We built a geometric model of a navigation system, with no preoperative image acquisition, to simulate the stages of the acquisition process. Random positions of each optical reflector center and anatomic acquisition point were generated within a sphere of predefined diameter. Based on the virtual geometric model and navigation process, we obtained 30,000 simulations using the Monte Carlo statistical method then computed the variability of the anatomic reference frames used to guide the bone cuts. Rotational variability (α, β, γ) of the femoral and tibial landmarks reflected implant positioning errors in flexion-extension, valgus-varus, and rotation, respectively., Results: Taking into account the uncertainties pertaining to the 3D infrared optical measurement system and to anatomic point acquisition, the femoral and tibial landmarks exhibited maximal alpha (flexion-extension), beta (valgus-varus), and gamma (axial rotation) errors of 1.65° (0.9°); 1.51° (0,98°), and 2.37° (3.84°), respectively. Variability of the infrared optical measurement system had no significant influence on femoro-tibial alignment angles., Conclusion: The results of a Monte Carlo simulation indicate a certain level of vulnerability of navigation systems for guiding position in rotation, contrasting with robustness for guiding sagittal and coronal alignments., Level of Evidence: Level IV., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Background and Objective: The use of a pneumatic tourniquet can induce muscular and neurological complications in the operated limb. The genesis of these injuries could involve an ischaemia/reperfusion phenomenon and a compression under the cuff. We evaluated effects of an antioxidant, acetylcysteine and ischaemic preconditioning on the rhabdomyolysis and postoperative pain following a knee ligamentoplasty using a pneumatic tourniquet., Methods: We included 31 patients scheduled for a knee ligamentoplasty randomly assigned in three groups (control, acetylcysteine 1200 mg the day before and 600 mg at the operative day, ischaemic preconditioning)., Results: There was a moderate rise in myoglobin and creatinine phosphokinase with no significant difference between the three groups. The muscular functional parameters were similar in all the groups. However, the morphine consumption within the first 48 h was smaller in the treatment groups (0.22 +/- 0.31 mg kg-1 and 0.22 +/- 0.23 mg kg-1 in the preconditioning and antioxidant groups, respectively) than in the control group (0.47 +/- 0.33 mg kg-1, P <0.05)., Conclusions: Acetylcysteine and ischaemic preconditioning do not decrease the extent of rhabdomyolysis related to the use of a pneumatic tourniquet and do not improve the postoperative muscle recovery. On the other hand, they allow a significant reduction in the postoperative morphine consumption.

The host-mediated in vitro/in vivo assay system was used to evaluate the tumorigenic potential of the aromatic nitro compound 2-nitroanisole (2-NA). After intraperitoneal administration of the compound, resident macrophages were recovered by peritoneal lavage from treated and untreated mice and cultured in soft agar. 2-NA was shown to be carcinogenic, and the tumorigenic potential was evaluated. Additionally, by establishment of a transformed peritoneal macrophage cell line, the underlying molecular mechanism of 2-NA's carcinogenic effect was studied.

Prostaglandin D synthase (L-PGDS) is a major glycosylated polypeptide in cerebrospinal fluid (CSF). The overexpression of L-PGDS in inflamed bovine mammary glands indicates its role as biomarker. No diagnostic tool for the quantitative detection of L-PGDS in cows has been reported. Immunometric ELISA tests might help to identify inflamed bovine tissue. The isolation of pure bovine L-PGDS, which is required for the generation of monoclonal antibodies, is an important prerequisite for a diagnostic ELISA test. Our goal was to identify a suitable technique to generate pure L-PGDS from bovine substrates. In the present study a two-step method for the purification of bovine CSF using ceramic hydroxyapatite chromatography followed by size exclusion chromatography is described. Subsequently, the identification of bovine L-PGDS was demonstrated by Western blot analysis and the high grade of the pure product was shown by 2-D PAGE. The yield of purified L-PGDS was 6.8 mg/l bovine CSF. L-PGDS from bovine CSF is shown to consist of multiple isoforms identical in molecular mass and pI values to those in previously described secretions of inflamed bovine mammary glands. In addition, the method was successfully applied to the purification of L-PGDS from human CSF.

12-O-retinoylphorbol-13-acetate (RPA), an incomplete tumor promoter of the phorbol ester type and protein kinase C (PKC) activator, consists of two characteristic structural elements: the phorbol body and the retinoyl ester chain. Therefore, possible binding of the incomplete tumor promoter RPA to the human transport protein retinol-binding-protein (RBP) has been examined by molecular modeling methods and experimental binding studies. The calculated prediction of binding properties was primarily based on a comparative geometrical approach. It was shown that the beta-ionone-ring of RPA was not altered within the binding pocket of RBP (molecular modeling) compared to retinoic acid (X-ray crystallographic data). The torsion angle C5'-C6'-C7'-C8', determining the conformation of the RBP-beta-ionone-ring relative to the isoprene tail, is rotated by 42 degrees for RPA compared to retinol and to retinoic acid, respectively. Combining all the results from force field calculations, MD simulations and geometrical comparisons, the conclusion could be drawn that RPA should be able to bind to RBP. This interaction should be less strong than that with its natural ligand retinol or with retinoic acid. This prediction was proven experimentally. RPA was able to compete with retinoic acid for binding at RBP in human plasma. The binding properties were investigated using 3H-labeled retinoic acid in homologous and heterologous competition studies in a one-dimensional native polyacrylamide gel electrophoresis system. An approximately 2000-fold weaker binding of RPA to RBP as compared to retinoic acid was determined experimentally, confirming the prediction of the molecular modeling approach. The characteristic behaviour of RPA as an incomplete promoter, due to possible binding to PKC and RBP, is discussed.

The successful separation of beta-lactoglobulin from other bovine whey proteins was performed by ceramic hydroxyapatite chromatography with a fluoride ion gradient in phosphate buffer as displacement agent. The method was applied to acid whey originating from milk of healthy as well as of mastitic cows. beta-Lactoglobulin was completely eluted in one peak at a fluoride concentration of about 0.6 mol/l. The purity of beta-lactoglobulin in this fraction was at least 96% if whey from healthy milk was processed. Co-eluted contaminants are traces of immunoglobulin G, serum albumin and lactoferrin. In case of mastitic whey the proportion of beta-lactoglobulin is diminished as the amounts of immunglobulin G, serum albumin and lactoferrin are increased within this fraction. Size exclusion chromatography on Superdex 75 pg effectively removed contaminants resulting in a purity for beta-lactoglobulin from normal whey of approximately 99%. The yield of beta-lactoglobulin from physiological whey was 50-55% referring to the fraction highly enriched with beta-lactoglobulin by hydroxyapatite chromatography. In case of mastitic milk the higher amounts of contaminants were also removed successfully by size exclusion chromatography.

A direct and an indirect antibody enzyme-linked immunosorbent assay for duck yolk IgY and duck serum IgY was developed and tested on egg yolk and serum of ducks vaccinated with Riemerella anatipestifer (Ra). Tests were performed either with primary antibodies labelled with horseradish peroxidase or with alkaline phosphatase-labelled secondary antibodies reacting with specifically bound rabbit anti-duck IgY antibodies, respectively. Ra-specific IgYs in egg yolk from three ducks increased rapidly at day 8 after the first of two vaccinations. In two ducks, the IgY titre persisted on a high plateau for 3 months. The concentration of Ra-specific IgYs in the serum of the progeny of vaccinees decreased between day 3 and day 10 after hatching. The fraction of total IgYs decreased less but also significantly. It was shown that antibodies were vertically transmitted and therefore protect offspring against Ra infection at least during the first week after hatching. The test design with anti-IgY rabbit antibodies is further suitable to detect other specific antibodies if respective antigens were fixed on solid phases.

Concentrations of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), polychlorinated biphenyls (PCBs), and organochlorine (OC) pesticides were measured in unsuccessfully hatched eggs of three different kinds of predatory birds: 3 eggs of sparrowhawks, 7 eggs of hobbies, and 16 eggs of goshawks collected in the German region of Berlin-Brandenburg. Using toxic equivalency factors for birds, eggs of hobbies contained mean concentrations of 478 pg TEQ/g fat and 551 pg TEQ/g fat contributed by PCDD/Fs and coplanar PCBs, respectively. For sparrowhawks the respective TCDD equivalents were 424 and 1278 pg/g fat; those for goshawks were 211 and 935 pg/g fat. The mean value of the summed concentrations of the PCB congeners 28, 52, 101, 138, 153, and 180 amounted to 11 microg/g fat in eggs of hobbies, 9.5 microg/g fat for sparrowhawks, and 16.1 microg/g fat for goshawks. Of the analyzed organochlorine pesticides the concentration of p,p'-DDE was highest (up to 273 microg/g); the concentration of the other OCs, with the exception of methoxychlor in eggs of goshawks (highest level of 10.6 microg/g fat), were negligible. In all cases the calculated toxicity of the coplanar PCBs surpassed the toxicity of the PCDD/Fs, at least by a factor of two. Due to the higher concentration of the coplanar PCB 77 compared to PCB 126 in eggs of hobbies, it is concluded that its metabolic excretion in this species is much lower than in sparrowhawks and goshawks.

The whey protein pattern of milk from animals affected by mastitic inflammation was resolved by two-dimensional gel electrophoresis (2D-PAGE) and compared to milk from unaffected cows. Inflammation caused the appearance of four spots aligned at a molecular weight level of 26 kDa and over a pH-region of 5.0 to 6.4. The spots excised from 2D gels were treated with chymotrypsin and the resulting peptides analyzed by MALDI-TOF mass spectrometry and RP-HPLC. All four spots yielded highly similar chymotryptic peptide mass fingerprints as well as chromatographic peak patterns. A database search could identify the four spots as isoforms of the bovine prostaglandin D synthase (PGD-S). In one of the isoforms a defined cysteine residue was shown to be oxidized to a sulfonic acid.

12-O-Tetradecanoyl-phorbol-13-acetate (TPA) has been shown to induce a broad variety of morphological and metabolic changes in mouse skin. These include gross changes in cell growth as well as subtle changes regarding gene expression. Changes in the induction of specific proteins have been described, but it is not yet clear, if there exists a strict causal correlation between the induction of expression of specific proteins and the phenomenon of tumor promotion. At least some of these proteins might also be correlated with an accompanying hyperproliferative effect. This has been shown for protein p10, a 10 kDa protein not found in untreated adult mouse epidermis. The expression of this protein can be induced de novo after the topical administration of TPA. It is also shown, that p10 is expressed in untreated neonatal murine epidermis. Due to the induction of this protein by irritation, hyperproliferation-causing tumor promoters and due to the observable abundance in neonatal epidermis, this protein seems not strictly related to tumor promotion, but rather to proliferation and differentiation.

Analysis of protein secretion was performed for a macrophage-like cell line, which was established from the peritoneal cells of NMRI mice treated with 17 alpha-ethinylestradiol. The protein secretion pattern was investigated by computerized analysis of high resolution two-dimensional gel electrophoresis (2-D PAGE) and compared to that of control macrophages, intraperitoneally activated by bacterial lipopolysaccharide. The transformed cells encode a number of low molecular weight proteins (10-20 kDa), which were not observed in control cells under identical experimental conditions. In conclusion the transformation of peritoneal macrophages by 17 alpha-ethinylestradiol leads to an upregulation of polypeptides, in particular of low molecular weight proteins. A high similarity between the induced low molecular weight protein secretion by macrophages of 17 alpha-ethinylestradiol-treated and that of 2,3,7,8-tetrabromodibenzo-p-dioxin-treated mice was found.

Systematic screening of different organs has shown that induction of particular proteins by 12-0-tetradecanoyl-phorbol-13-acetate is specific for mouse skin. Different compartments of skin showed all the same expression. For experimental reasons we focused the analysis performed thereafter on the epidermis. As an end- point of mouse skin tumorigenesis papilloma and carcinoma were also screened for changes in protein expression. We focused our efforts on the characterization and identification of selected, de novo expressed proteins induced by various tumor promoters in murine epidermis. The analysis presented here consists of functional characterization. Having found differences in protein expression in TPA-treated murine epidermis compared with acetone-treated control mice, we then tried to correlate the expression to the signal transduction pathway which the tumor promoter exerts its activity through. For this reason we screened protein expression patterns after topical administration of mechanistically differently acting tumor promoters, which were inflammatives not bearing tumor promoting activity anti-promoters or a combination of tumor promoters with modulators of tumor promotion. The epidermis of untreated newborn mice served as a model for high cellular proliferation and differentiation. Alterations were compared with changes observed after tumor promoter application.

Treatment of murine epidermis with the tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA) shows characteristic and significant changes in protein expression analyzed by 2D PAGE, compared to that of acetone-treated mouse epidermis. Of the seven de novo expressed proteins in TPA treated murine epidermis, one is a 44 kDa protein (p44) located nearby actin, and six proteins are in the low-molecular range between 10-20 kDa (p10, pY, pCa, p1, p2 and p3). Interestingly, the incomplete promoting and inflammative hyperplasiogen 12-O-retinoylphorbol-13-acetate (RPA) and the non-promoting but inflammative and hyperplasiogenic calcium-ionophore A23187 induced the same pattern of proteins observed in the pidermis of mice treated with TPA, with minor quantitative differences. In all cases, p10 expression was quantitative the most abundant. Partial sequencing of this protein has led to the conclusion that it is a novel protein with no such sequences in the database comparisons using FASTA and TFASTA computer programs of Genetics Computer Group. The data presented here do not strictly support the functional role of de novo induced proteins to tumor promotion, but show a causal relationship to hyperplasiogenic potency of TPA, RPA and A23187.

In 1996 forty-nine eels were caught from different locations along the rivers Oder (Hohenwutzen and Schwedt) and from 11 locations along the Havel river. They were analysed for PCDDs/PCDFs and the coplanar PCBs (PCB 77, PCB 126, PCB 169). Their contribution to 2,3,7,8-TCDD equivalents were estimated. In case of PCDDs/PCDFs these amounted between 1.8 and 15.2 pg/g fat (mean 6.1 pg/g; median 5.2 pg/g), in case of coplanar PCBs between 2.4 and 170.5 pg/g fat (mean 47.7 pg/g; median 36.1 pg/g). Lakes which are associated with but not directly located at the main stream of the river Havel contributed much less to contamination of the eels than the river segments situated in the more urbanised or industrialised sites along the main river.

The usefulness of soluble markers in indirectly differentiating inflamed quarters from healthy quarters of 30 Holstein Friesian cows was tested over a follow-up period of 20 weeks. In comparison to N-acetyl-beta-D-glucosaminidase (NAGase), lactate dehydrogenase (LDH), Na+, K+ and Cl- the somatic cell counts (SCC) were determined in foremilk samples. The soluble parameters were measured by means of an autoanalyzer system (Beckman CX5CE) equipped with solid state ion electrodes. Due to different types of inflammatory reactions it is considered to be advantageous to perform two tests in parallel for assessing the mammary gland status. Cell-damaging processes should best be detected by measuring elevated LDH activity, whereas the impairment of the blood-udder barrier is recorded by the respective elevation of the indicators Na+ and Cl-. Among all variables, SCC included, the soluble parameters showed the least dispersions, whereas those of SCC were the most unfavourable in this respect. Moreover, NAGase/LDH and Na+/Cl- among all variables had the highest correlation coefficients of 0.85 and 0.86, respectively. Threshold values as decision limits are proposed. Additionally, an inter-quarter comparison and evaluation improved the diagnostic value of each indirect mastitis parameter analyzed. By means of discriminant analysis, it is shown that the distribution of inflammation within the udders of this herd was unequal.

The protein secretion patterns in a macrophage-like cell line (CBrD), established from the peritoneal cells of NMRI mice treated with the dioxin analog 2,3,7,8-tetrabromodibenzo-p-dioxin (TBrDD), were analyzed by high resolution two-dimensional gel electrophoresis (2-D PAGE), and compared to the pattern of proteins secreted by control macrophages which were intraperitoneally activated by bacterial lipopolysaccharide. The most striking alterations were observed in the low molecular range. The transformed cells encode a number of low molecular mass proteins (10-20 kDa) which were not detected in control cells under identical experimental conditions. The protein pattern with respect to isoelectric point, molecular weight, optical density (OD) and area of the spot (in mm2) has been depicted by computer analysis in relation to a standardized spot outline and the spot's background (in OD). It is concluded that the transformation of murine peritoneal macrophages by TBrDD leads to an upregulation of proteins, in particular of low-molecular-weight proteins.

The guideline no. 206 for testing of chemicals of the Organization for Economic Cooperation and Development (OECD) comprising an avian reproduction test using the Japanese quail (Coturnix coturnix japonica; Termminck and Schlegel 1849) as pair-hold test organisms has been applied in a version that reduced the treatment period to 6 weeks without any pretreatment. In the present study bis(tri-n-butyltin)oxide, C.A. No. 56-35-9 (tributyltin oxide, TBTO) was examined by five participants in an interlaboratory comparison test. A comparable regimen of dosing was performed by all participants starting either with 24 or 60 mg/kg TBTO in the feed and ending with 150 or 375 mg/kg. Within this dose range no signs of toxicity in adults were observed. Substance-related effects however were obvious with regard to egg production, fertility, hatching success, and survival of 14 day-old chicks. A clear dose dependency was given regarding effects on egg weight and on hatchability. The no-observed-effect concentrations for these two parameters was 60 mg/kg TBTO, characterizing these parameters as the most sensitive in this investigation. With the presented set of test parameters further aspects of subchronic toxicity in adults and chicks can be assessed as well as the validity of the performed test. Comparing the results for most test parameters consistency is obvious, thus confirming the applicability of the presented test guideline.

We have developed a host-mediated assay system for detection of the transforming activity of chemical carcinogens on peritoneal macrophages, directly, as well as indirectly acting carcinogenic substances administered intraperitoneally to NMRI mice could be examined in this way. Resident macrophages were recovered by peritoneal lavage from treated and untreated mice and cultured in soft agar. After 5-6 days normal and transformed cells could be distinguished. Statistical analysis comparing cells from 2, 3, 7, 8-tetrachlorodibenzo-dioxin (TCDD)-treated animals with those from control mice proved that the test is positive at least on a significance level of 5%, using the t-test. TCDD revealed a cell-transforming potential that showed a dose-dependent response in this host-mediated assay. The co-carcinogenic activity of TCDD was established in experiments with diphenylhydantoin. Low doses of diphenylhydantoin which did not exhibit any transforming potential in our system gained a high oncogenic potential by the simultaneous administration of low doses of TCDD, which also had no transforming activity. We have compared the cell transforming potential of TCDD with its bromo analog TBrDD. The cell transforming potential of TCDD is 7 times that of TBrDD. We have succeeded in establishing a permanent cell lined from mice treated with TBrDD. The oncogenicity of this cell line was tested in athymic nu/nu mice. Animals treated subcutaneously with these cells (1 x 10(6) cells) developed tumors at the injection site. Using monospecific antibodies to tumor necrosis factor alpha (TNF-alpha), we have found that TCDD stimulates the secretion of TNF-alpha. The experimental data reported here lead to the conclusion that TCDD has a carcinogenic as well as a co-carcinogenic activity and has the property to induce TNF-alpha.

Xiphophorine fish from wild populations are insusceptible to develop neoplasia. In contrast, certain backcrosses of Xiphophorus maculatus (platyfish) with Xiphophorus helleri (swordtail) as the recurrent parent, produce offspring that develop neoplasia in a Mendelian fashion. We concentrated our research on melanoma. The starting signal for the development of melanoma comes from an accessory v-erbB-related oncogene, x-erbB*, which is highly homologous to the human EGF receptor gene, and is part of a platyfish-specific tumor gene-complex designated as Tu. Normally, the platyfish is protected from its own Tu by Tu-specific regulatory gene systems. The swordtail has neither evolved the Tu-complex nor the regulatory gene systems. Therefore, the backcross procedure dismantles the regulatory gene systems thus permitting Tu-directed melanoma formation. x-erbB* derived from the platyfish, together with the swordtail-derived oncogenes src, sis, pdgf-r, ras, myc, erbA are expressed or overexpressed in the melanoma, and inositol lipid turnover is considerably elevated. x-src and inositol lipid turnover have also been found elevated in the healthy tissues (eg brain) of the tumourous fish. To construct a fish strain which is highly susceptible to mutagenic carcinogens, we introduced a particular regulatory gene, ie an oncosuppressor gene (Bs), into the genome of the animals developing Mendelian inherited melanoma, by introgressive breeding. Bs prevents the strain from germ line-inherited melanoma but, following carcinogen-induced impairment in a somatic cell gives rise to the Tu-directed development of clonal melanoma in a particular fish, x-src activity and inositol lipid turnover are elevated in the tumor but, in contrast to the animals bearing the inherited melanoma, are not elevated in the brain. Promoting carcinogens (tumor promoters) do not induce melanoma in this strain. Similarly, in order to breed a fish strain susceptible to tumor promoters, we introduced a regulatory gene (the oncostatic gene g, golden) coding for a pre-transformational arrest of pigment cell differentiation in the stem cell stage of the fish that develop the Mendelian inherited melanoma. The new strain is incapable of developing melanoma. Its x-src activity and inositol lipid turnover is elevated in the brain indicating that the biochemical processes which were found to be correlated with the hereditary melanoma formation, are running in the new strain without the occurrence of melanoma. Following treatment with tumor promoters that overcome the arrest of pigment cell differentiation, melanoma develops within a very short latent period.

Xiphophorine fish from wild populations are insusceptible of developing neoplasia. In contrast, certain backcrosses of Xiphophorus maculatus (platyfish) with Xiphophorus helleri (swordtail) as the recurrent parent produce offspring that develop neoplasia in a Mendelian fashion. We concentrated our research on melanoma. To construct a fish strain which is highly susceptible to mutagenic carcinogens, a particular regulatory gene, ie an oncosuppressor gene (Bs), was introduced into the fish developing the Mendelian inherited melanoma by introgression. Bs prevents the progeny from developing melanoma. However, Bs can be impaired by carcinogen-induced somatic mutation which gives rise to the development of clonal melanoma. Activity of the oncogene x-src (measured on pp60x-src kinase activity) and inositol lipid turnover is elevated in the tumor but, in contrast to the animals bearing the inherited melanoma, not in the brain. Tumor promoters do not induce melanoma in this strain. Similarly, in order to breed a fish strain which is highly susceptible to tumor promoters we introduced a regulatory gene, for instance an oncostatic gene (g) coding for a pretransformational arrest of pigment cell differentiation in the stem cell stage of the fish that develop the Mendelian inherited melanoma. The new strain is incapable of developing melanoma. Its x-src kinase activity and inositol lipid turnover is elevated in the brain, indicating that the biochemical processes which were found to be correlated with the hereditary melanoma formation, operate without the occurrence of melanoma. Following treatment of these animals with tumor promoters, melanoma develops within a very short latent period. Our tester strain can discriminate between tumor-initiating and tumor-promoting activities of agents of unknown carcinogenic potential.

The effect of di(2-ethylhexyl)phthalate (DEHP) on diethylnitrosamine (DEN)-initiated preneoplastic liver lesions with expression of gamma-glutamyltranspeptidase (GGTase) and loss of adenosine triphosphatase (ATPase) as well as alterations of hepatic carbohydrate metabolism in male and female Sprague-Dawley rats have been investigated. Two treatment schedules have been compared with respect to their sensitivity by the histochemical demonstration of preneoplastic islands and by the biochemical determination of alterations in enzyme activities of liver homogenates and of serum, the last indicating hepatotoxicity. For initiation, a single dose of DEN was given, followed by treatment with various doses of DEHP given three times weekly by gavage for 7 or 11 consecutive weeks. As histochemical enzyme markers, the expression of positive GGTase as well as the deficiency in ATPase were used for identification of liver foci. The weanling female rats (protocol A) were found to be more sensitive to the carcinogenic effect of DEN in view of foci incidence than the mature male rats which underwent partial hepatectomy prior to DEN application. The administration of 200 mg DEHP/kg body wt increased the incidence of ATPase-deficient foci in both male and female rats; however, concentrations of 1000 and 2000 mg DEHP/kg decreased the incidence of liver foci. The number of foci with expression of GGTase was only slightly increased in female rats following a DEHP concentration of 50 mg/kg, and 200 mg/kg body wt. DEHP alone did not induce preneoplastic lesions that could be identified by these two markers. Biochemical investigations indicate that DEHP alters the metabolic pattern in liver. An increase of the NADP-linked enzymes glucose-6-phosphate dehydrogenase (G6PDH), malic enzyme, extra-mitochondrial ICDH as well as an enhancement of NAD-dependent alpha-G3PDH and lactate dehydrogenase were found following DEHP administration. On the other hand the glycolytic enzymes pyruvate kinase (PK) and enolase as well as the gluconeogenetic enzyme fructose-1,6-bisphosphatase (FBPase) were significantly reduced. In protocol B (male rats) the reactions of PK, FBPase and malic enzyme were more altered after DEHP exposure than in protocol A, while the activity of G6PDH was more increased in protocol A. Most enzymes being involved in the carbohydrate metabolism are influenced by DEHP in a dose-dependent manner. There was no increase in serum FBPase activity in both male and female rats after DEHP treatment but a reduction of glutamate-oxalate-transaminase and glutamate-pyruvate-transaminase activities was observed.(ABSTRACT TRUNCATED AT 400 WORDS)

We have developed a host-mediated assay system for the detection of the transforming action of chemical carcinogens on peritoneal macrophages. Directly as well as indirectly acting carcinogenic substances administered intraperitoneally to NMRI mice could be examined in this way. Resident macrophages were recovered by peritoneal lavage from treated and untreated mice and were cultured in soft agar. After 5-6 days normal and transformed cells could be distinguished. Statistical analysis comparing cells, for example, from alpha-naphthylamine or diphenylhydantoin-treated animals with those from control mice proved that the test is positive at least on a significance level of 5% using the t-test. Further substances revealing a cell-transformation potential were benzene, benz(a)pyrene, 2,3,7,8-tetrachlorodibenzodioxin, N-nitrosodimethylamine, ethidium bromide, aflatoxin B1,N-methyl-N-nitrosourea, 1-methyl-3-nitro-1-nitrosoguanidine, 2-naphthylamine, dieldrin, suramin and trichloroethylene. A weak transforming potential was found for chlorambucil as well as for tetrachloroethylene. With toluene or azidothymidine no cell transformation could be observed. Several immortal cell lines could be established form NMRI mice treated with alpha-naphthylamine or N-methyl-N-nitrosourea. Athymic nu/nu mice injected subcutaneously with these cells developed tumors, establishing the oncogenic potential of these cell lines.

The German Chemicals Act requires that chemicals are tested for behavioral toxicity at stage 2 of the testing procedure, i.e. if more than 1000 annual tons are produced. For this purpose a guideline was developed according to which data on behavioral toxicity are to be collected, which are based on cageside observations during longterm exposure. The protocol covers outer appearance, as well as motor, sensory, autonomic and central nervous system functions. Data are to be reported in tabular form and should be evaluated by taking all aspects of the toxicological profile into account.

The synthesis of rRNA in cultured human liver cells grown in the presence of L-triiodothyronine (1.6 x 10(-5)M) was measured by means of polyacrylamide gel electrophoresis using a double labelling technique. The rate of accumulation of rRNA in the cytoplasm as well as the turnover of rRNA in ribosomes remained unchanged in comparison to control cells regardless of growth stage or cell population density. L-triiodothyronine, however, induced an increased synthesis of rpreRNA when added in the early growth phase of sparse cultures. This induction was time dependent and occurred temporarily, but it was never followed by a similar accumulation of mature rRNA. Therefore, in this system two control mechanisms counteracting each other were effective: the increased rpreRNA synthesis indicates a transcriptional control, and the wastage of odd rpreRNA, however, a post-transcriptional control.

The content of ribosomal DNA in mice liver at the beginning as well as near the end of the hematopoietic period was measured by RNA/DAN-hybridization in solution. At both stages the amount of ribosomal DNA was the same and comparable to that of postnatal liver.

Three rat liver foci bioassays have been compared with respect to their sensitivity by the histochemical demonstration of preneoplastic foci, and by the biochemical determination of alterations in enzyme activities of serum indicating hepatotoxicity. We studied the initiation/promotion schedules according to Oesterle and Deml (A), and according to Pereira (B, Broad Spectrum Protocol), and the initiation/selection protocol according to Tatematsu et al. (C), with diethylnitrosamine (DEN), given as a single initiating dose of 10 and 30 mg/kg body wt respectively. With all schedules Sprague-Dawley rats, either females, 3 weeks old (A), or males, 6 weeks old (B, C) were used. For promotion polychlorinated biphenyls (A) or phenobarbital (B) were administered. Selection was performed with 2-acetylaminofluorene (C). The rats in schemes (B) and (C) underwent partial hepatectomy one day prior to initiation. The number and total area of foci deficient in adenosine-5'-triphosphatase (ATPase) and positive in gamma-glutamyltranspeptidase (GGTase) was evaluated. In the complete schedule with 30 mg of DEN in system (A) foci incidence exceeded that of the other systems by about 7-fold (ATPase) and 2-fold (GGTase) respectively. The lower dose of DEN and all control experiments resulted in a respective lower foci yield. With scheme (C), but not with schemes (A) and (B), e.g. serum fructose-1.6-bisphosphatase and alkaline phosphatase were increased, suggesting liver cell damage. Thus tested with DEN, scheme (A) is most sensitive and causes a low impairment of animals' welfare.