Myocardial degeneration was also seen in one mid-dose male. Adverse effects were also observed in the top dose groups of all other amines. Decreased body weights associated with diminished food intake were generally seen. Slight increases in packed cell volume, haemoglobin concentration and thrombocytes occurred with cadaverine. With spermidine, decreased plasma creatinine, calcium and inorganic phosphate were observed and decreased potassium levels with cadaverine. The no-observed-adverse-effect level was 2000 ppm (180 mg/kg body weight/day) for tyramine, cadaverine and putrescine.

INTRODUCTION

Many foods of vegetable or animal origin contain a great number of mono-, di- and polyamines, the so-called biogenic ar~:ines. These amines are organic bases with a low molecular weight with an aliphatic, aromatic or heterocyclic structure. Some of these amines are formed n~Lturally by metabolic processes, whereas others are formed by enzymatic decarboxylation from amino acid!s by microbial activity (Maga, 1978; Smith, 1980-81). The level in foods varies widely, from very low to thousands of parts per million (Askar and Treptow, 1986; Bos et al., 1986; Haldsz et al., 1994; Maga, 1978; Smith, 1980-81, Ten Brink et al., 1990).]In the last decades, increasing attention has been paid to these amines, because it became apparent that they may exert biological activities of a diverse nature and play an important role in many physiological functions. For instance, certain biogenic amines are important in the regulation of DNA, RNA and protein synthesis, and probably also in the stabilization of membranes (Gahl and Pitot, 1981; Smith, 1980-81; Tabor and Rosenthal, 1956; Tabor and Tabor, 1964). 

 There is a large body of data on the pharmacological effects of biogenic amines in humans, especially with respect to histamine and tyramine. However, despite the potentially high biological activity of many biogenic amines and their occurrence in many foods, sometimes at a rather high level, no toxicity data are available that allow the establishment of a no-observed-adverse-effect level (NOAEL) in laboratory animals. Therefore, it is also not possible to calculate an acceptable daily intake (ADI) for man. n. For this reason, a programme was initiated to investigate the oral toxicity of five of these amines which are quantitatively most important in foods, namely tyramine, cadaverine, putrescine, spermine and spermidine. In addition, acute iv studies were conducted to examine the influence of the five biogenic amines on blood pressure. The results of acute and subacute studies with these amines in Wistar rats are reported here. 

MATERIALS AND METHODS 

P-(2-aminoethyl)-phenol-hydrochloride (tyramine) (mol. wt 173.64 g/tool, Ref. No. 8373, purity 99%) was obtained from E. Merck AG (Darmstadt, Germany ). N - ( 3 - Aminopropyl ) - 1,4 - butanedia - mine-trichloride (spermidine) (tool. wt 254.63 g/mol, Ref. No. 85580, purity > 99%), N,N'-bis(3- aminopropyl ) - 1, 4 - butanediamine-tetrahydrochloride (spermine) (tool. wt 348.19 g/mol, Ref. No. 85610, purity > 99%), 1,4-diaminobutane-dihydrochloride (putrescine) (tool. wt 161.08 g/tool, Ref. No. 32810, purity > 99%), and 1,5-diaminopentanedihydrochloride (cadaverine) (tool. wt 175.10 g/tool, Ref. No. 33220, purity > 99%) were all obtained from Fluka AG (Buchs, Switzerland). 

Animals and maintenance 

Wistar-derived SPF-bred rats (Cpb:WU; Wistar random or Bor:WISW) were obtained from TNO Central Institute for the Breeding of Laboratory Animals, Zeist, The Netherlands or from F. Winkelmann Institute for the Breeding of Laboratory Animals GmbH & Co. KG, Borchen, Germany. They were housed conventionally under barrier conditions, in suspended stainless-steel cages fitted with wiremesh floor and front, two rats per cage in the acute studies and five rats per cage in the subacute studies. The room temperature was maintained at 22 + 3°C and the relative humidity at 55 + 15%. Artificial light was provided continuously, for 12 hr/day from 07.30 hr until 19.30 hr. The number of air changes was about 10/hr.

Diets

The rats used in the acute studies were fed the Institute's basal diet, which is a cereal-based open formula diet (Rutten and de Groot, 1992). For the subacute studies the Institute's basal diet or a purified diet was used. The percentage composition of the purified diet was as follows: casein 20; bL-methionine 0.2; wheat starch 64.8; cellulose (solka floc) 5; mineral mixture (Jones Foster) 4; vitamin B preparation 0.2; vitamin ADEK preparation 0.4; choline chloride (50%) 0.4; soyabean oil 5. In the first and second study with sperrnidine, the Institute's basal diet was used. Because the level of some biogenic amines in the basal diet (on analysis, in mg/kg/diet: tyramine 92, spermidine 43, spermine 19, putrescine 102 and cadaverine 174) was considerably higher than in purified diet (on analysis, in mg/kg/diet: spermidine 0.5 and putrescine 0.1), the subacute studies with tyramine, spermine, putrescine and cadaverine were carried out with purified diet. 

Haematology and clinical chemistry. Blood samples were collected from the tip of the tail of all rats early in wk 5 and examined for haemoglobin concentration, packed cell volume and erythrocyte, leucocyte and thrombocyte counts (Sysmex K-1000 Haematology Analyzer, Toa Medical Electronics Co, Ltd, Kobe, Japar~), differential leucocyte count (blood smears stained according to Pappenheim) and prothrombin time (Normotest, Nyegaard & Co. A/S, Oslo, Norway). Whole blood taken from all rats after overnight fasting in wk 5 was examined for glucose (Boehringer Glucoquant No. 245-178; Boehringer Mannheim GmbH, Mannheim, Germany). Blood samples taken from the abdominal aorta of all rats at autopsy were centrifuged at 1250 g for 15 min and then analysed for alkaline phosphatase (ALP), aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), 7-glutamyl transferase, total protein, albumin, urea, creatinine, total bilirubin, calcium and inorganic phosphate (Cobas-Bio Centrifugal Analyzer, Hofmann-La Roche, Basle, Switzerland), chloride (Chloro Counter, Marius, Utrecht, The Netherlands) and sodium and potassium (Electrolyte-2 Analyzer, Beckman Instruments, Brea, CA, USA). Urinalysis. In wk 5 all rats were deprived of water for 24 hr and of food for 16 hr. Urine was collected during the last 16 hr of the deprivation period and its volume (calibrated tubes) and density (refractometer; Bellingham and Stanley, London, UK) were measured. Pathology. The rats were killed in wk 5 or 6 by exsanguination from the abdominal aorta while under light ether anaesthesia, and a thorough autopsy was performed. 

 Towards the end of the study all survivors in the top-dose group showed abdominal distension and slight paralysis of the hind legs. Growth rate, food intake and water intake were considerably decreased in the top-dose group, but not in the lower dose groups (Table 3). Systolic blood pressure was decreased in males of the top-dose group and also in the female that survived in this group (Table 3).In males of the top-dose group, haemoglobin concentration, packed cell volume, mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) were decreased and mean corpuscular haemoglobin concentration (MCHC), prothrombin time and thrombocyte count were increased (Table 4). Packed cell volume, MCV and MCHC showed the opposite changes in the surviving female in this group. Clinical chemistry (Table 5) revealed statistically significant increases in plasma ASAT and ALAT activities and in inorganic phosphate and sodium levels at 5000 ppm in both sexes, and in ALP activity in females. Plasma calcium level was decreased in males of the 5000 ppm group and in the surviving female of this group, while plasma potassium concentration was decreased in males of the mid- and high-dose group groups. Fasting glucose and urea levels were increased in the top-dose males, whereas albumin and chloride levels were decreased in the surviving female of this group. In the high-dose group, the concentration test showed a considerable increase in urinary volume accompanied by a considerable decrease in density (Table 8). The relative weights of the testes, brain, thyroid, adrenals, spleen and heart were increased and that of the liver decreased in males of the 5000 ppm group (Table 6). 

cortical region. The liver had a slight to moderate decrease in glycoge:n content. In the heart, slight to moderate focal myocardial degeneration accompanied by mononuclear cell infiltration occurred in the high-dose rats and in one mid-dose male. The rats that died showed severe depletion of the white pulp in the spleen and, in the thymus, acute involution characterized by los:s of normal architecture of cortex and medulla, and severe necrosis of lymphocytes. Putrescine. Mean body weights, food intake and food efficiency were slightly decreased with 5000 ppm in both sexes, although the differences from the controls were statistically significant for body weights in females and for food intake in males (Table 3). Plasma alanine amino-transferase activity was slightly increased in females of the 5000 ppm group (Table 5). In the 5000 ppm group, the relative weight of the brain was significantly increased in females (Table 6). Cadaverine. Mean body weights, food intake and food efficiency were slightly decreased with 10,000 ppm, although the differences were not always statistically significant (Table 3). Haematology (Table 4) revealed slight increases in packed cell volume and MCHC in males and in haemoglobin concentration and thrombocyte count in females at 10,000 ppm. Clinical chemistry (Table 5) revealed slightly increased plasma ASAT and ALAT activities in males at 10,000 ppm, while the plasma potassium level was slightly decreased in this group. The relative weights of the testes and brain were significantly increased in the 10,000 ppm group. Relative liver weight was decreased in the top-dose males (Table 6).

A single iv administration of sperm±dine (3 mg/kg), spermine (1 mg/kg), putrescine (10mg/kg) and cadaverine (30 mg/kg) induced a reduction in systolic and diastolic blood pressure. A transient fall in blood pressure after iv injection of spermine (0.15 M/kg; 52 mg/kg) was reported by Tabor and Rosenthal (1956). In the oral feeding studies no decreases in systolic blood pressure were observed, except for a slight decrease in systolic blood pressure in the top-dose spermine group; this was most probably due to malfunction of the kidneys, as evidenced by a marked decrease in concentrating ability and histopathological renal changes. The increased relative weights of the adrenals and the myocardial degeneration in the top-dose spermine group may have been related to the decrease in blood pressure. Myocardial degeneration conceivably results in decreased cardiac output, and a reduced blood pressure may lead to chronically increased adrenomedullary production of pressor substances, resulting in an increased adrenal weight. The very different dynamics of systemic exposure following iv administration v. dietary oral administration is probably a major reason for seeing only a slight effect with spermine and for not seeing any effect with the other biogenic amines. Decreased body weights associated with diminished food intake and sometimes also with decreased food efficiency were observed in the top-dose group of all five biogenic amines examined. The adverse effects on body weight were most pronounced with spermine, followed by putrescine, cadaverine, spermidine and tyramine.  At the next lower levels (e.g. 1000 ppm for spermidine and 2000 ppm for the other four amines), these effects did not occur.

 Putrescine fed to newly hatched Japanese quail at a dietary level of 2000 ppm for 9 days also had no apparent effect on body weight (Blonz and Olcott, 1978); furthermore, a combination of cadaverine (1710 ppm), putrescine (910ppm), tyramine (910ppm) and histamine (540 ppm) fed to rats for 32 days had no effects on growth or nitrogen utilization (Haaland and Njaa, 1989). These previous results support our own findings that levels of up to 1000 or 2000 ppm of the five biogenic amines examined do not influence growth rate. In a study with 7-day-old chickens, however, it appeared that supplementation of tyramine or cadaverine at fairly low levels (33 and 82 ppm, respectively) to basal diets with a high concentration of biogenic amines from fish meal resulted in a decrease in growth, food intake and food efficiency, suggesting a high sensitivity of young chickens to biogenic amines (Bakker, 1994). The mortality, weight loss, decreased food and water intake, aggressive behaviour and convulsions observed in animals fed 10,000 ppm spermine in the first week are probably due to the toxicity of sperrnine at this high level. After this level had been decreased to 5000 ppm, there was some stabilization in the condition of the surviving animals in this group; however, at the end of the study they showed paralysis of the hind legs. The increased prothrombin time, blood glucose level, plasma ALAT and ALP activities and the relatively low plasma albumin level in high-dose males could not be related to liver damage and may have been due to the considerable decrease in food intake in these rats (Levin et al., 1993; Oishi et al., 1979; Schwartz et al., 1973).

 Spermine was the only biogenic amine that showed treatment-related decreases in red blood cell variables in males of the high-dose group. These changes in red blood cell picture might be the result of the marked disturbed renal function observed in the high-dose spermine group. The increases in packed cell volume, haemoglobin concentration and thrombocyte counts and the decrease in MCHC value with 10,000 ppm cadaverine occurred in only one of the sexes and the differences from the controls were slight. However, at present there is no explanation for these findings and therefore they are regarded as treatment related. In the high-dose groups with tyramine, spermidine, putrescine and cadaverine, changes occurred in one or more of the plasma epzymes related to the liver (ALP, ASAT and ALAT) together with increases or decreases in the relative weight of the liver; with spermidine a decrease in total plasma protein levels in females was also observed. As all these changes were slight and no treatment-related histopathological changes were observed in the liver, they are considered to be a reflection of changes in the metabolic function of the liver rather than indications of a hepatotoxic effect of these four biogenic amines. With spermine, the decrease in liver weight observed in males of the high-dose group may be related to the decreased glycogen content in the liver seen on microscopic examination. The top-dose spermine treated rats clearly showed impaired renal function, as evidenced by increased urinary volume and decreased density in the concentration test, which was accompanied by histopathological changes of the kidneys, increased plasma levels of urea and creatinine and changes in plasma electrolytes. This clearly indicates that spermine is nephrotoxic to the rat.

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