Use of E mu-PIM-1 transgenic mice short-term in vivo carcinogenicity testing: lymphoma induction by benzo[a]pyrene, but not by TPA

E mu-pim-1 transgenic mice are predisposed to develop lymphomas. Due to their low spontaneous tumour incidence and their increased sensitivity towards the lymphomagen ethylnitrosourea these mice may present an interesting model for short-term carcinogenicity testing. Here, we report on the further exploration of this transgenic mouse model with two additional carcinogens known to have, among others, the lymphohaematopoietic system as target, i.e. benzo[a]pyrene (B[a]P) and 12-O-tetradecanoylphorbol-13-acetate (TPA). B[a]P, given three times a week (by gavage) for 13 weeks at 4.3, 13 or 39 mg/kg body weight, resulted in a dose-related increase in lymphomas up to a 90% incidence in E(mu)-pim-1 mice during the observation period of 40 weeks. B[a]P also induced tumours of the forestomach within this observation period, though at a lower incidence and apparently equally effective in wildtype and transgenic mice. TPA, on the other hand, was unable to induce lymphomas (or tumours in any other organ) in either transgenic or wildtype animals within the observation period of 44 weeks, when applied dermally at the maximum tolerated dose of 3 microg/mouse, twice a week for 35 weeks. Molecular analysis showed that B[a]P-induced lymphomas in transgenic mice were of T-cell origin, 80% of which had elevated levels of c-myc expression. None of the lymphomas had increased N-myc expression and mutation analysis of the ras-gene family revealed a K-ras mutation in only one out of eight tumours investigated. Also, none of the lymphomas showed aberrant expression of p53 as determined by immunohistochemistry. It is concluded that the E mu-pim-1 mouse model will not be very suitable for short-term carcinogenicity testing in general: only genotoxic chemicals that have the lymphohaematopoietic system as target for carcinogenesis in wild- type mice, appear to be efficiently identified.      

Quantification of platelet-bound immunoglobulins of different class and subclass using radiolabeled monoclonal antibodies: assay conditions and clinical application

Quantification of platelet-bound immunoglobulins (PBIg) with radiolabelled murine monoclonal antibodies (mAbs) has been described only for IgG so far. Here we describe some modifications of this mAb radioimmunoassay (MARIA) and show that by using a panel of radiolabelled specific mAbs it is possible to quantify not only PBIgG but also PBIgG subclasses and PBIgM. Analysis by gel filtration showed that all anti-IgG and anti-IgG-subclass mAbs bound to their respective antigens in a ratio of about 1:1. However, the binding ratio for the anti-IgM Mab could not be established. There was a good correlation between the antibody-density per platelet as determined with the anti-IgG mAb and determined as the sum of the IgG molecules of different subclass per platelet (r = 0.90). Platelet fragments did not interfere in the assay. 89 normal healthy controls had 140 IgG molecules per platelet and bound 269 anti-IgM molecules per platelet (geometric means). In a study on the detection of PBIg in 147 thrombocytopenic patients, it appeared that the MARIA had a sensitivity of 61% and a specificity of 45% for the diagnosis of idiopathic thrombocytopenic purpura (ITP). Both in ITP and in secondary thrombocytopenia (STP), PBIgG1 and PBIgG3 were found more frequently (60% and 61%, respectively) than PBIgG2 and PBIgG4 (13% and 9%, respectively). There was no relation between the amount of total PBIgG or PBIgM and the platelet count in either ITP or STP. Also, if IgG antibodies of only one subclass were found, there was no relation between the severity of the thrombocytopenia and the amount of PBIgG. By applying the MARIA, it is possible to quantify PBIgG, all four PBIgG-subclasses and PBIgM in ITP and STP in a reliable way.     

Biliary excretion of mercury compounds.

The disappearance of ²⁰³Hg from the plasma of rats and its excretion into bile was quantitated for 2 hr after the iv administration of 0.03, 0.1, 0.3, 1.0, and 3.0 mg Hg/kg as ²⁰³mercuric chloride. The concentration of ²⁰³Hg in the bile was usually about 0.66 that in the plasma. The concentration of ²⁰³Hg in the liver was 1.8–3.4 times higher than that in the plasma, and the bile concentration was about three times lower than that in the plasma. Methyl mercuric chloride was given to rats at dosages of 0.1, 0.3, 1.0, and 3.0 mg Hg/kg, iv. The concentration of ²⁰³Hg in bile averaged about nine times higher than that in the plasma, the liver concentration was about 25-fold higher than that in the plasma and the bile concentration about 0.33 that in the liver. Thus the radioactivity associated with either mercuric chloride or methyl mercury were not highly concentrated in bile as are some other heavy metals. Over a 2-hr period, regardless of the dose or the form of Hg administered, less than 0.5% of the dose was excreted into the bile. The effect of 4 days pretreatment with phenobarbital, spironolactone, pregnenolone-16-carbonitrile (PCN), and 3-methylcholanthrene on the biliary excretion of mercuric chloride and methyl mercury was also measured. PCN was the most effective, doubling the amount of ²⁰³Hg excreted into the bile.     

Effect of spironolactone on the distribution of mercury.

The distribution and biliary excretion of ²⁰³HgCl₂ (0.3 mg Hg/kg) iv was measured in rats treated with spironolactone (SP, 75 mg/kg, ip) for various time intervals. SP had its greatest effect when administered as a single dose 15 min before HgCl₂. SP decreased the concentration of ²⁰³Hg in the plasma from 1.5 to 0.05 μg/ml, while it increased the blood concentration from 1.5 to 5 μg/ml. This treatment increased the content of Hg in the lung 12, heart 6, spleen 3, brain 3, muscle 2, stomach 1.7, and liver 1.5 times control, had no effect on the concentration of ²⁰³Hg in the intestine, bone, and testes, and markedly decreased the amount in the kidney to 10% of controls. Biliary excretion of Hg was not increased. When SP was administered 90 min or 3 hr before administration of the ²⁰³HgCl₂, qualitatively similar but less dramatic effects on the distribution of Hg were obtained. SP administered 15 min after HgCl₂ administration had a similar effect on the distribution of Hg as when administered 30 min before HgCl₂, with the exception that the concentration of Hg in the kidney was not decreased. The two major metabolic products of SP, canrenone and thioacetic acid, were also given to determine their effect on Hg distribution. Canrenone had no effect while thioacetic acid produced an effect similar to that produced by SP. It appears that the alteration in the distribution of Hg after SP treatment is due to the sulfur portion of the molecule. It seems likely that the sulfur moiety complexes the Hg; this complex distributes in the body similar to organic mercurial compounds, which in comparison to inorganic mercurials, reach a lower concentration in the plasma and kidney and a higher concentration in the blood and other tissues. The decrease in the concentration of Hg in the kidney produced by SP is probably responsible for the decreased toxicity of Hg after SP treatment.     

Methicillin-resistant Staphylococcus aureus in pig farming

We conducted a study among a group of 26 regional pig farmers to determine the methicillin-resistant Staphylococcus aureus prevalence rate and found it was >760 times greater than the rate of patients admitted to Dutch hospitals. While spa-type t108 is apparently a more widespread clone among pig farmers and their environment, we did find other spa-types.