Whole-body autoradiographic study on the distribution of 3H-cryptolepine in mice.

The distribution of 3H-cryptolepine in pigmented and albino mice and in pregnant mice on day 16 of gestation was studied by whole-body autoradiography, after a single intravenous injection. The labelled compound rapidly left the blood and was localized in most tissues, except for the central nervous system where no radioactivity could be observed. The most pronounced decrease of concentration occurred between 1 and 4 hours postinjection. A relatively high and lasting level of radioactivity was found in organs with rapid cell proliferation and in the liver. The most notable and prolonged retention, however, was found in the adrenal medulla and in the melanin-containing tissues of the eye (up to 8 days after administration--the longest survival time). In the pregnant animals, the activity in fetal tissues was much lower than in the maternal. It has previously been found that cryptolepine produces blood pressure-lowering effects, which may be due to the marked localization in the adrenal medulla. The retention in the melanin-containing tissues of the eye (both in adults and fetuses) may indicate a potential risk for the induction of adverse effects in pigmented tissues, especially after chronic administration of the drug.

Differential distribution and placental transport of 2- and 3-t-[methyl-14C]butyl-4-hydroxyanisole (BHA) in pregnant mice.

The placental transport and localization in fetal and maternal tissues of 14C-BHA isomers, 2-t-[methyl-14C]butyl-4-hydroxyanisole (2-BHA) and 3-t-[methyl-14C]butyl-4-hydroxyanisole (3-BHA), were studied in pregnant mice by whole-body autoradiography techniques. BHA isomers were given (iv 50 microCi/100 g as a tracer dose) to pregnant mice at Day 11 (organogenesis) and Day 18 (postorganogenesis) of gestation. Peak levels of radioactivity occurred in various tissues 1-4 hr after iv administration of both isomers. 3-BHA and its metabolites have a higher affinity to fatty tissues and livers of pregnant mice. The concentration of radiocarbon in maternal liver and brown fat following treatment with 14C-3-BHA was much higher than the radioactivity concentration in the corresponding tissues of mothers treated with 2-BHA. On the other hand, the fetal concentration of radioactivity was higher in animals treated with 2-BHA than in those treated with 3-BHA. The radioactivity derived from both isomers accumulated in the fetal gastrointestinal tract. In both groups the radioactivity accumulated in the maternal nasal cavity and mucosa and the gastrointestinal contents. At 24 hr after treatment, retention of radioactivity in maternal lungs, amniotic fluid, and fetal gastrointestinal tissues was observed. Results from this study indicate that there are differences in the magnitude and extent of placental transport of 3-BHA and 2-BHA. Differences also exist in maternal organ uptake and radioactivity distribution of both isomers. Findings from this study are consistent with pharmacological differences existing between the isomers.

Computer-assisted quantification and image processing of whole-body autoradiograms.

A computerized image-processing system especially adapted for analysis of whole-body autoradiograms has been developed. It consists of commercially available standard components, including a black-and-white video camera, a microcomputer, and graphics equipment. The lower performance of the hardware has been compensated for by more flexible software. When the system was calibrated, special attention was paid to local variations in the measuring system in different parts of the picture. Utility programs for the manipulation of contrast, pseudocoloring, and image enhancement, etc., are available. Some programs have been especially designed to comply with specific problems and demands related to different autoradiographic applications. A program displaying the density histogram for an area of interest is particularly useful for the quantitation of whole-body autoradiograms. It allows the operator to select interactively a range of densities. Image elements (pixels) corresponding to the densities in this range are shown in red on the monitor, and their average true density is calculated. This procedure permits the marking and analysis of delicate structures on autoradiograms. Other programs allow a picture, stored in memory, to be rotated or translated, and two pictures to be superimposed for comparison. Various applications of using image analyses in whole-body autoradiography are presented and illustrated.

Selective incorporation of thiouracil into murine metastatic melanomas.

The uptake and retention of 14C-thiouracil and 125I-thiouracil in small lung metastases of B16 murine melanoma was studied in beige mice injected intravenously with melanoma cells. By impulse counting of excised tumor and organ pieces, a high concentration of radioactivity was found in the lung metastases, as compared to normal tissues. The highest tumor/organ concentration ratios appeared 24 h after injection of the radiolabeled thiouracil. A separate autoradiographic study on the disposition of 14C-thiouracil in mice with melanoma metastases confirmed the impulse counting results and also showed the absence of any other site of retention of radioactivity except for hair follicles and to some extent the thyroid. The selective uptake of 14C- and 125I-thiouracil in melanomas depends on their acceptance as false melanin precursors, making them specific markers for growing melanin. The results indicate that radiolabeled thiouracil may be useful for clinical diagnosis and, possibly, therapy of malignant melanotic melanomas.

Rate of incorporation of [3H]thymidine in various tissues of the mouse. A basis for the evaluation of genotoxic effects of chemicals.

[3H]Thymidine has been extensively used as a selective precursor to DNA in studies on the kinetics of cell proliferation. We have become interested in measuring early inhibition of the DNA synthesis in various organs of intact animals for detecting genotoxic properties of chemicals. Such experiments should, for convenience and to achieve a large capacity, be performed in the simplest way possible. The present paper deals with some practical aspects on the use of [3H]thymidine in vivo. [6-3H]Thymidine was injected intraperitoneally in mice and the uptake of radioactivity was evaluated by using whole-body autoradiography and liquid scintillation spectrometry. Autoradiograms of sections washed with trichloroacetic acid and methanol were compared with those subjected only to freeze-drying. Liquid scintillation counting was performed of total, non-volatile, acid-insoluble and DNA-associated radioactivities. A rapid increase of the [3H]thymidine incorporation was seen during the first hour after the injection. Further prolongation of the survival time did not result in any significant increase of the incorporated radioactivity. Moreover, there were only slight differences between the autoradiograms from extracted and non-extracted sections. Radioactivities associated with DNA closely correlated to those representing acid-insoluble material, indicating that acid-insoluble radioactivity provides a good estimate of the [3H]thymidine incorporation into DNA.

Tissue-specific inhibition of [3H]thymidine incorporation into DNA by carcinogenic N-nitrosamines.

The N-nitrosamines N-nitrosodimethylamine (DMN), N'-nitrosonornicotine (NNN) and 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were injected intraperitoneally 24 h before sacrifice in F344 rats and C57BL mice in doses of 297 mumoles/kg b.w. and 148 mumoles/kg b.w., respectively. 2 h before sacrifice, the animals were given an intraperitoneal injection of [3H]thymidine. The results showed that the examined N-nitrosamines inhibited the incorporation of [3H]thymidine into DNA in a few tissues of the rats and the mice. The results indicated that the N-nitrosamines exerted a tissue-specific inhibition of the [3H]thymidine incorporation in the tissues reported to be involved in the biotransformation of these substances. The observed inhibitory effects on the incorporation of [3H]thymidine by DMN, NNN and NNK were also correlated to a considerable extent to the reported sites of carcinogenicity. The present study indicates that measurements of [3H]thymidine incorporation into DNA in various tissues of experimental animals is a useful short-term bioassay to evaluate the potential tissue-specific carcinogenicity of the N-nitrosamines. The method may also be useful as a complement to other short-term in vivo tests in the screening of potential genotoxicity of several other chemicals.

Whole-body autoradiography using 11C with double-tracer applications.

This paper reports that it is possible to obtain good autoradiographic pictures of a 11C-labelled compound ([11C]methionine) in spite of the short half-life of 20 min by modifying the whole-body autoradiographic (WBA) technique. Double radionuclide autoradiography was performed by utilizing the great difference between the half-lives of 11C and 14C or 35S. The first exposure resulted entirely from 11C, and the second exposure the day after from 14C or 35S, respectively. Satisfactory blackening of the film was obtained by a mean radioactivity of 1.6 X 10(6) disintegrations/cm2 during the exposure. The 11C-WBA technique apparently provides a useful tool in distribution studies on animals, which is valuable in the interpretation of positron emission tomography images of the same 11C-compound.

The in vivo uptake of tritiated thymidine as a potential short-term test of toxic effects of polycyclic aromatic hydrocarbons in different organs.

A rapid in vivo test for toxicity is described where the test substance is allowed to distribute and metabolize in the intact mouse. Quantitative data are provided on the effect of 3 different polycyclic aromatic hydrocarbons on the DNA turnover in various organs, measured as the incorporation of tritiated thymidine. In accordance with previously reported carcinogenic potencies, 7,12-dimethylbenz[alpha]anthracene (DMBA) was more potent in inhibiting the thymidine incorporation than benzo[alpha]pyrene (B[alpha]P). The inhibitory effect was most pronounced in spleen, lung, pancreas, small intestine and kidney, resulting in a decrease of incorporated activity with up to 80%. There was no evidence for the existence of specific target organs for DMBAs effects on thymidine incorporation as indicated by an inhibitory action in all organs studied. A decreased thymidine incorporation after administration of DMBA could also be demonstrated with whole-body autoradiography. The inhibitory effect of B[alpha]P was most pronounced in thymus, spleen, small intestine and testis, the average decrease of incorporated activity being more than 40% after 48 h. Contrary to the wide action of the above mentioned polycyclic aromatic hydrocarbons, an equimolar dose of anthracene lacked significant effects on the various organs.

Selective affinity of dimethyl sulphoxide (DMSO) and 2-amino-4-phenylsulphonylbenzenesulphonamide (NSD 3004) for the large intestinal mucosa of mice.

Whole-body autoradiography of 14C-labelled dimethyl sulphoxide and 35S-labelled 2-amino-4-phenylsulphonylbenzenesulphonamide revealed a selective accumulation of labelled substance in the large intestinal mucosa of mice. The mechanism of accumulation is discussed with regard to previous results on sulphone-containing compounds with tissue-specific distribution patterns.

A new melanoma seeker for possible clinical use: selective accumulation of radiolabelled thiouracil.

In a previous report we have shown that a few substances, especially thiouracil, are incorporated as false precursors into melanin during its synthesis. In the present investigation, we have intensified our studies on the incorporation of thiouracil into melanotic melanomas. Firstly, the distribution and retention of both 14C- and 35S-labelled thiouracil in mice with transplanted melanomas were studied. A high and selective accumulation was found in the melanotic tumours. The concentration in the rest of the body was low, with the exception of the thyroid gland. Secondly, melanoma-bearing mice were given increasing doses of thiouracil, and cultured melanoma cells were exposed to different concentrations of thiouracil, to investigate the relation between dose and uptake in melanomas and melanoma cells, respectively. A relatively linear increase in uptake with dose was found, indicating that the melanin incorporation of thiouracil is non-saturable up to subtoxic levels.

A whole body autoradiographic study on the distribution of 14C-labelled di-(2-ethylhexyl)phthalate in mice.

Di-(2-ethylhexyl)phthalate (DEHP), a plasticizer used for polyvinylchloride polymers, has been reported to leach from blood transfusion bags and the plastic material in hemodialysis units into the blood. Phthalate esters are known to be hepatotoxic and teratogenic in experimental animals. Reports on the distribution and metabolism of DEHP indicate that the compound in largely excreted from the body within a few days. In the present investigation the distribution and tissue retention after administration of [14C]DEHP (carbonyl-14C or 2-ethylhexyl-1-14c) was studied in pregnant and non-pregnant mice with whole body autoradiography. Initially a high activity was observed in the brown fat, liver, gall bladder, intestinal contents, kidney and urinary bladder. Pretreatment with DEHP, phenobarbital sodium or 3-methylcholanthrene caused a relative increase of the activity in the brown fat, indicating that induced metabolic conversion of DEHP leads to an increased deposition of radioactivity in brown fat. After administration of DEHP (carbonyl-14C), but not DEHP (2-ethylhexyl-1-14C),marked retention was observed in the skin, cartilage and tendons. The mechanism responsible for the slow accumulation in these connective tissues is not known. In the early embryo a high concentration was observed in the neuroepithelium. This pronounced uptake may be correlated to the DEHP-induced malformations exencephaly and spina bifida observed in mice.

Incorporation of thiouracil and some related compounds into growing melanin.

Several drugs, mainly polycyclic amines, are accumulated in melanin-containing tissues. They are bound to preformed melanin (both in vivo and in vitro). Thiouracil is accumulated into melanin according to another principle: It is incorporated as a false precursor during melanin formation. It is thus taken up only in growing melanin, e.g. in the eye of pigmented mouse foetuses or in melanomas. The acceptance of a foreign substance during the formation of a foetal tissue seems theoretically important. We are also interested in the practical viewpoint of using false melanin precursors as selective melanoma seekers. Some related substances are therefore compared with respect to incorporation into growing melanin. The thiouracil uptake in the ocular melanin of a 5 day old mouse was 276 times higher than that of a 3 month old mouse based on weight units of melanin. Thiourea is incorporated in growing melanin as well but also binds slightly to preformed melanin. Uracil and fluorouracil showed no specific uptake into growing melanin. It thus seems as if the sulfur is essential for the incorporation into the melanin polymer. 35S-thiouracil and 2-thio(2-14C)urcal showed the same high uptake, indicating that at least part of the uracil moiety is incorporated together with the sulfur. There seems to be a relation between the property to be incorporated into melanin and the tyrostatic activity. Both in the formation of melanin and thyroid hormones, tyrosine is the physiological precursor and both reactions are catalyzed by oxidizing enzymes. Properly labelled thiouracil derivatives seem to be promising melanoma seekers for diagnostic and radiotherapeutic purposes.

Long-term fate of [14C]nicotine in the mouse: retention in the bronchi, melanin-containing tissues and urinary bladder wall.

N-methyl-14C and 2'-14C-labelled nicotine were used for whole-body autoradiographic distribution studies on C57BL- and NMRI-mice. Radioactivity was retained in the melanin-containing tissues, in the bronchial walls, and in the urinary bladder wall, up to 1 month after administration. The activity levels in the bronchi decreased faster if [2'(14)C] nicotine was used. Quantitative measurements of the retention of the 2 14C-labelled nicotine preparations confirmed the autoradiographic findings. It is proposed that nicotine is N-demthylated in the bronchial mucosa, the off-coming methyl group being incorporated into the cell constituents of the mucosa. Thin-layer chromatographic studies showed that no nicotine was present in the lungs after 24 h. In melanin, however, only unmetabolized nicotine was found from 4 h on. Some reactive nicotine metabolites may be responsible for the retention in the urinary bladder wall. Also in the full-term fetuses radioactivity accumulated in the pigmented eyes and in the respiratory tract. The accumulation and long-term retention of nicotine in the melanin-containing structures might accelerate the development of drug-induced or senile changes in these tissues. The retention in the urinary bladder wall persisted even after rinsing. This may indicate an accumulatory mechanism worth considering in the pathogenesis of urinary bladder cancer.

Distribution of an 125I-labelled chloroquine analogue in a pregnant macaca monkey.

Whole body autoradiography of a pregnant monkey (Macaca irus) of late gestation was performed 72 h after an intravenous injection of the 125I-labelled chloroquine analogue 4-(3-dimethylaminopropylamino)-7-iodoquinoline (DAPQ). The overall distribution pattern in the monkey was similar to that which was earlier observed in rodents. A few species differences, however, were found in the monkey as compared to the rodents: a high accumulation in the inner part of the adrenal cortex, a high level in the central nervous system, and generally a higher retention in the tissues. The accumulation in the adrenal cortex may be of significance for the cortisone-like effects of the 4-aminoquinolines in rheumatoid arthritis and allied conditions. The fact that no accumulation was found in the adrenal cortex of mice and rats indicates that these species may not be appropriate in studies on the mechanisms involved in the anti-inflammatory action of the 4-aminoquinolines. As was earlier observed in small rodents the melanin containing structures accumulated the drug. In both the mother and the fetus a high concentration was thus seen in the uveal tract of the eye, in the inner ear (in the stria vascularis of the cochlea and the planum semilunatum of the ampullae) and in the hair follicles. This accumulation can be related to reported disturbances--also transplacentally induced--in vision and hearing.

Distribution of local anesthetics: accumulation in some endocrine polypeptide-hormone producing cell systems.

1 After the injection of labelled procaine and lidocaine in mice, the location and concentration of radioactivity was demonstrated by autoradiographical methods.2 An accumulation in some endocrine cells such as the pancreatic islets, the hypophysis, the adrenal medulla and certain cells of the thyroid (probably representing the calcitonin-producing parafollicular cells) was shown.3 After the injection of [(14)C]-procaine in chicks, an accumulation of radioactivity was observed in the ultimobranchial gland (which produces calcitonin in birds), but not in the thyroid.4 Radioactivity was also shown to be strongly concentrated in structures containing melanin, such as the pigment of the eye, skin and hair and in some organs involved in the metabolism and excretion of these drugs.