Immunotoxicological investigation of subacute combined exposure with low doses of Pb, Hg and Cd in rats.

Detectable interactions between NOEL (No Observed Effect Level) doses of Pb, Hg and Cd in general toxicological, hematological, and immune function parameters were investigated. The metals (Pb-acetate, 20 mg/kg; HgCl2, 0.40 mg/kg; CdCl2, 1.61 mg/kg) were combined. First, the rats received the combination Pb + Hg + Cd for 4 weeks per os. Significant difference vs. control was found only in the weight of lung and popliteal lymph node (PLN). The Pb + Hg and Pb + Cd combinations significantly decreased the PLN to 100 g body weight and PLN to brain weight ratio, and Pb+Hg also decreased the relative adrenal weight. After 12 weeks treatment with the same doses, effects on the thymus, kidney, and adrenal weights in the Pb + Hg, and thymus weight in the Pb + Cd, combination were seen. Pb + Cd also affected the white and red blood cell count and hematocrit. Combined with Hg or Cd, NOEL dose Pb showed toxicity, indicating that exposure limits may be inefficient in combined exposure situations.

Immuno- and neurotoxicological investigation of combined subacute exposure with the carbamate pesticide propoxur and cadmium in rats.

In the present study, the effects of subchronic per os exposures to cadmium chloride (CdCl(2)), and a carbamate insecticide, propoxur (Pr), were investigated in male Wistar rats on general toxicological (body weight gain, relative organ weights) haematological (RBC, WBC, Ht, MCV, cell content of the femoral bone marrow) immune function (plaque forming cell (PFC) assay, delayed type hypersensitivity (DTH) reaction) and neurotoxicological (spontaneous and stimulus-evoked cortical activity, nerve conduction velocity) parameters. The animals were treated for 4, 8 and 12 weeks with 6.43 mg/kg CdCl(2), 8.51 mg/kg Pr, or with a combination of 6.43 mg/kg CdCl(2)+0.851 mg/kg Pr or 8.51 mg/kg Pr+1.61 mg/kg CdCl(2). Cadmium exposure affected the relative thymus, liver, and adrenal weight, RBC count, haematocrit and MCV, and there was an increase in nerve conduction velocity and a decrease in the cortical evoked potential latency. Pr induced a decrease in thymus weight, had some effect on the liver weight but none on the electrophysiological parameters. A significant interaction between Cd and Pr was detected by the following parameters: RBC, Ht, PFC, and nerve conduction velocity. The results indicate that combined exposures in humans may result in a shift in the apparent detection limits and/or in the LOEL of the single substances. The latter raises the necessity to reconsider exposure limits in situations where the risk of combined exposure is high.

A study on geno- and immunotoxicological effects of subacute propoxur and pirimicarb exposure in rats.

A 28-day oral exposure with 8.51, 3.40, and 0.851 mg/kg propoxur (PR) and 4.67, 1.87, and 0.467 mg/kg pirimicarb (PI) was performed in male Wistar rats, and the occurrence of numerical and structural chromosome aberrations and the changes in certain immune function parameters (plaque-forming cell (PFC) assay, delayed-type hypersensitivity reaction) and in some basic toxicological (body weight gain and weights of brain, thymus, lung, heart, liver, spleen, kidneys, adrenals, and popliteal lymph node) and hematological (white blood cells, red blood cells, hematocrit (Ht), mean cell volume of red blood cells (MCV) cell content of the femoral bone marrow) parameters were investigated. The high dose of PR increased the relative liver weight and the cell content of femoral bone marrow, and all three doses increased Ht and MCV. The applied doses of PI decreased the relative adrenal weight in a dose-dependent manner, and its highest dose increased the relative liver weight. Among the immune function parameters tested, PFC content of the spleen was decreased by high-dose PR and elevated by high-dose PI, whereas the maximum and the time course of the delayed-type hypersensitivity reaction showed no changes in this dose range. In the genotoxicological investigations only the high PR dose increased the number of numerical, but not the structural, chromosome aberrations. In addition to the changes in relative adrenal weight following PI treatment, the PFC assay showed the highest sensitivity for detection of the 4-week exposure with these carbamates. On the basis of our results, the immunotoxicological approach seems to have the same (PR) or higher (PI) sensitivity in early detection of the repeated low-dose exposure by these carbamates compared to the genotoxicological approach.

Immunotoxicological investigations on rats treated subacutely with dimethoate, As3+ and Hg2+ in combination.

Effects of combined exposure with dimethoate (DM), HgCl2 (Hg), and NaAsO2 (As) were investigated following a 28 - day oral exposure in male Wistar rats. In preliminary experiments, the LOEL (Lowest Observed Effect Level) and NOEL (Non Observed Effect Level) doses of the substances were determined using the same experimental system [determination of body weight gain, organ weights of brain, thymus, heart, lung, kidneys, adrenals, spleen, testicles, popliteal lymph node, white blood cell (WBC) and red blood cell (RBC) count, haematocrit (Ht), mean cell volume (MCV) of RBCs, cell content of the femoral bone marrow, IgM-plaque forming cell (PFC) content of the spleen, delayed type hypersensitivity (DTH) reaction] and animal strain. In the combination studies, LOEL dose of DM (28.2 mg/kg) was combined with NOEL doses of the heavy metals ( HgCl2 = 0.40 mg/kg, NaAsO2 = 3.33 mg/kg), and vice versa (DM = 7.04 mg/kg, HgCl2 = 3.20 mg/kg, NaAsO2 = 13.3 mg/kg). In the DM-Hg combinations, significant alterations were found versus the corresponding high- dose internal control in the body weight gain, relative liver and kidney weights, and in the PFC response. When DM was combined with As, interactions were indicated by changes of relative liver weight, MCV value, and the PFC content of the spleen. These results support the theory that the interactions between pesticides and heavy metals may modify the toxic effects of the single substances, and may also change the functional detection limits of the exposure. The changes in the functional detection limits, if they occur, can lead to false-positive and false-negative results in human epidemiological studies.

Detection of the effects of repeated dose combined propoxur and heavy metal exposure by measurement of certain toxicological, haematological and immune function parameters in rats.

In the present study, an immunotoxicity test system, containing general toxicological (body weight gain, organ weights), haematological (WBC,RBC, Ht, mean cell volume of the RBCs, cell content of the femoral bone marrow), and immune function (PFC assay, DTH reaction) investigations, was used for detection the effects of a 4 weeks repeated low dose combined oral exposure of male Wistar rats with propoxur and the heavy metals arsenic or mercury. Two doses of the compounds were used: a higher one (the lowest dose which resulted in significant change of at least one parameter examined in previous dose-effect experiments), and a lower one (the highest dose which proved to be non-effective). The applied doses were: 8.51 and 0.851 mg kg(-1) of propoxur, 13.3 and 3.33 mg kg(-1) of NaAsO(2), and 3.20 and 0.40 mg kg(-1) of HgCl(2). In the combination treatment, the high dose of propoxur was combined with the low dose of arsenic or mercury, and the high doses of each heavy metals were combined with the low dose of propoxur. The main finding of this study was that some of the combinations significantly altered the relative weight of liver, adrenals and kidneys, related to both the untreated and the high dose internal control. Among the immune functions examined, only the PFC content of the spleen showed a trend of changes in certain combinations versus the corresponding high dose control. According to the present results, combined exposure with propoxur and the heavy metals examined can modify the detection limit of the single compounds and/or may alter their toxic effects.

Immunotoxicological investigation of subacute combined exposure by permethrin and the heavy metals arsenic(III) and mercury(II) in rats.

Effects of combined 28 days of oral exposure to the insecticide Permethrin (Pe), alone or in combination with arsenic-III (As) or Hg-II (Hg), were investigated on certain toxicological (body weight, organ weights), haematological (white blood cell (WBC) and red blood cell (RBC) counts, haematocrit (Ht), mean cell volume (MCV), cell content of the femoral bone marrow) and immune function (IgM-PFC, delayed-type hypersensitivity (DTH) reaction) parameters of male Wistar rats. Immunotoxic (H = high) and NOEL (L = low) doses of the three substances were determined in preliminary experiments under identical experimental conditions. In the present study, the immunotoxic dose of Pe (126 mg/kg) was combined with the NOEL dose of As (3.33 mg/kg) or Hg (0.40 mg/kg), and the NOEL dose of Pe (12.6 mg/kg) with the immunotoxic dose of As (13.3 mg/kg) or Hg (3.20 mg/kg). A separate group of animals, treated with the appropriate high dose component only, was used as internal control. Significant interactions were observed in the liver weight of the animals treated with Pe(H)-As(L) or As(H)-Pe(L), in the cell content of the femoral bone marrow in case of Pe(H)-As(L) and Pe(H)-Hg(L) combinations, as well as in the number of PFCs formed from 10(6) spleen cells in the Pe(H)-As(L) and in the maximum of DTH reaction in the Hg(H)-Pe(L) combination. The results show that combined exposures by the investigated substances modify the toxic (including immunotoxic) effects of the single compounds. These findings rise the probability that the interactions observed can also be present in human situations altering the health hazard of this three chemicals.

Neurotoxic states and their investigation--possibility for early detection of poisoning.

A highly important function of the nervous system (NS) is to process the information arriving to us from external, environmental sources. This capacity may be discontinued by chemicals restricting the plasticity of the NS, disturbing its adaptability to the ever changing environment. NS and endocrine system are profoundly bound together, changes, disruption of the different regulatory endocrine levels by toxicants may take part in the development of manyfold neural disfunction. The various endocrine levels (integrative, modulatory, regulatory, effector, target gland, etc.) affecting neurotoxicity and affected by neurotoxicity are discussed. The NS is protected against some toxic insults but is rather sensitive to other ones. By electrophysiological and psychophysiological methods, used as sensitive biomarkers the signs of functional disturbances of the NS can be proved both in human investigations and in animal experiments at an early stage of low concentration intoxications when clinical or general laboratory symptoms do not indicate yet the moderate poisoning.

Small subchronic doses of the pesticide dimethoate and/or cadmium and lead treatment causes disturbances in the chromosomes of young rats.

Small doses of Dimethoate (DM) and cadmium (Cd) which in themselves proved to be harmless in causing chromosome aberrations, potentiate each other's toxic effect concerning both numerical and structural aberrations caused in rat bone marrow cells. The toxic effect of lead (Pb) was not enhanced by DM.

Simultaneous geno- and immunotoxicological investigations for early detection of organophosphate toxicity in rats.

Detectability of toxic effects by repeated doses of dimethoate (DM) and methylparathion (MPT) were investigated by geno-and immunotoxicological methods in male Wistar rats following a 28-day oral exposure. In the dose range of 28.2, 14.1, and 7.04, and 7.04 mg/kg/day DM, the two higher doses decreased the body weight gain. The top dose increased the weight of liver, kidneys, and testicles; the white blood cell count; and the cell content of the femoral bone marrow. From immune function parameters measured [IgM-plaque forming cells (PFC) assay, delayed-type hypersensitivity (DTH) reaction] only the maximum of the DTH reaction decreased at the top dose. Of the MPT doses (0.872, 0.436, and 0.218 mg/kg/day) the two higher ones increased the liver weight, and a dose-dependent increase was found in the MCV value. No evaluable changes in the examined immune function parameters were observed. Both substances increased the number of numerical but not the structural chromosome aberrations at lower dose levels (the two larger doses of DM, and all the three doses of MPT) than those ones which caused changes in the examined immune function parameters. According to these results, the genotoxicological approach seems to be more sensitive for detection of repeated-dose oral toxicity of the investigated two organophosphates than the immunotoxicological one.

Simultaneous action of cypermethrin and two environmental pollutant metals, cadmium and lead, on bone marrow cell chromosomes of rats in subchronic administration.

The aim of the present study was to investigate the possible genotoxic effects, exerted by the pyrethroid cypermethrin and by either of the metals cadmium and lead alone or in combination, on bone marrow cell chromosomes in a subchronic experiment. Outbred male Wistar rats were treated per os for 4 weeks in a five-time per week schedule with 5.54, 11.08, or 22.16 mg/kg cypermethrin (1/100, 1/50, 1/25 LD50) alone, or in combination of 1/100 and 1/50 LD50 cypermethrin with 2.0 mg/kg cadmium chloride or 10 mg/kg lead acetate. On the day following the last treatment, the animals were sacrificed and bone marrow from the femur was prepared. Twenty metaphases from 10 animals per group were evaluated. The evaluation comprised the frequency of aberrant cells, the numerical and structural aberrations, and the alterations in relative organ weights. In the dosage used, cypermethrin and cadmium alone caused no significant increase in the chromosomal aberrations, and lead acetate caused an increase of the numerical aberrations only. Combination of cypermethrin and cadmium also failed to induce significant chromosomal effects. The cypermethrin + lead combination, however, induced a significant increase of structural chromosomal aberrations, predominantly of all acentric fragments. This lead to the conclusion that the simultaneous administration of lead and cypermethrin results in an enhanced genotoxic effect.

Immunotoxicological effects of repeated combined exposure by cypermethrin and the heavy metals lead and cadmium in rats.

The immunotoxic effect of a 28 days oral exposure by 55.4, 22.2, and 11.1 mg/kg cypermethrin (CY) was investigated in 4 weeks old male Wistar rats. The applied test system involved the determination of general toxicological parameters (body weight gain, organ weight of thymus, heart, lung, liver, spleen, kidneys, adrenals and the popliteal lymph node), haematological parameters (white blood cell count, red blood cell count, haematocrit, mean cell volume of red blood cells, cellularity of the femoral bone marrow), as well as immune function assays (splenic plaque forming cell assay, delayed type hypersensitivity reaction). The highest dose resulted in a significant increase of the relative liver weight, and all three doses resulted in (although inconsistent) changes in the haematocrit and MCV values. The maximum of DTH reaction decreased at all three doses. On combination of the highest CY dose with non-effective doses of lead or cadmium the immunotoxic effects of the former were modified. When immunotoxic doses of Cd or Pb were combined with the lowest CY dose, further interactions were observed on the examined parameters. The alterations of the immunotoxic effects of CY by simultaneous exposure with Cd or Pb, as described here, can lead to unexpected health consequences and/or can lead to false positive or negative results in human epidemiological studies.

Comparison of detection sensitivity of immuno- and genotoxicological effects of subacute cypermethrin and permethrin exposure in rats.

Immuno- and genotoxicological effects of a 28-day oral treatment by equitoxic (1/10, 1/25, 1/50 LD50) doses of cypermethrin (55.4, 22.2, and 11.1 mg/kg) and permethrin (125.7, 50.3, and 12.6 mg/kg) were compared on male Wistar rats. Humoral and cell-mediated immunity were investigated by PFC assay and delayed type hypersensitivity (DTH) reaction (footpad swelling assay), and the genotoxic effects were studied by structural and numerical chromosome aberrations in bone marrow cells. The experimental system also involved certain general toxicological (body weight gain, organ weights) and haematological [white blood cell (WBC), red blood cell (RBC), haematocrit (Ht) and cell content of the femoral bone marrow] investigations. Among the immune function assays, only DTH reaction decreased at the two higher cypermethrin (CY) doses. These doses also increased the number of numerical chromosome aberrations of the bone marrow cells but did not change the number of structural aberrations. All CY doses decreased the mean cell volume (MCV) of RBCs and the Ht value. The two higher doses also reduced the WBC count in the peripheral blood. Permethrin (PE), in the applied dose range, had no effect on the examined immune function parameters, but all three doses increased the number of numerical chromosome aberrations. A dose-dependent increase in the liver weight, decreased MCV value, and elevated cell content of the femoral bone marrow were also observed. Under these experimental conditions, examination of chromosome aberrations proved to be less sensitive in detection of exposure by cypermethrin than applied immune function assays did. Permethrin, on the contrary, increased the number of numeric aberrations at all dose levels but had no effect on the immune function parameters examined.

Electrophysiological biomarkers of an organophosphorous pesticide, dichlorvos.

The correlations between the relatively low level, chronic dichlorvos (DDVP) treatment and the early electrophysiological changes were investigated in three series of experiments. In the first series, male Wistar rats were orally given daily by gavage 0.98, 1.96 and 3.92 mg/kg of DDVP for 4, 8, or 12 weeks, and recordings were made at the end of each period. In the second one, the male and female rats of three consecutive generations were treated with the same doses, the investigated parameters were recorded at the age of 12 weeks of the male animals. In the third experiment, the doses were administered in different stages of development: during pregnancy, pregnancy + lactation, pregnancy + lactation + postweaning, and the electrophysiological markers were recorded also at the age of 12 weeks of male offspring. The analyzed elecrophysiological parameters were: electrocorticogram, sensoric cortical evoked potentials, conduction velocity and refractory periods of peripheral nerve. The data showed that the relatively low level dichlorvos exposure caused dose-, duration-, generation-, developmental stage-dependent and partly significant alterations in all the investigated electrophysiological parameters. The analyzed functional parameters proved to be sensitive biomarkers of the exposure as they were changed by the lower doses, while the brain cholinesterase activity was considerably inhibited only in the groups given the highest dose.

Immunotoxicological examination of repeated dose combined exposure by dimethoate and two heavy metals in rats.

The immunotoxicity of 28 days combined oral exposure by dimethoate (DM) and two heavy metals (Pb or Cd) was investigated in male Wistar rats. Immunotoxic and no-effect doses of DM (28.2 and 7.04 mg/kg) were combined with immunotoxic and no-effect doses of CdCl2 (6.43 and 1.61 mg/kg) or lead acetate (80.0 and 20.0 mg/kg) in such a way that the high dose of each substance was given in combination with the no-effect dose of the other. To examine the interactions of these agents, general toxicological (body weight gain, organ weights), haematological (absolute and differential WBC, RBC, MCV, Ht. cell content of the femoral bone marrow), and immune function (splenic PFC number. DTH reaction) parameters were measured. Treatment with the combination of Pb or Cd and DM did not result in a reduction of humoral (PFC) and cellular (DTH) immune responses, whereas treatment with the substances alone did result in immune suppression. This protecting effect can probably be attributed to an effect on the kinetics of the compounds tested rather than on the immune system itself. Further interactions were found in both combinations, DM-Cd and DM-Pb, in the body weight gain and in the relative liver weight; the DM-Pb combination also affected the relative thymus weight and the MCV value. These findings show that the immunotoxic effects of the investigated materials, including their detectability and health consequences, can be modified in case of combined exposure.

Epidemiological investigations and experimental model studies on exposure of pesticides.

Authors carried on epidemiological examinations on plant protection workers to find early symptoms (biomarkers) of moderate contamination by pesticides. Measurement of changes in cholinesterase and gamma glutamyltransferase enzyme activity did not reveal that people were affected. Determination of chromosome abnormalities from peripheral lymphocytes disclosed numerical aberrations at an early stage and lasted after cessation of work for several months. There were differences in the results among different groups of plant protection workers, as well as among those working in open field or in glasshouses. Women, who carried out horticultural work in glasshouses after spraying showed some chromosome abnormalities too. Experimental rats treated chronically with organophosphates exhibited changes in spontaneous and evoked brain electrical activity (ECoG). Concerning the peripheral nervous activity, conduction velocity became slower, absolute and relative refractory periods became longer. These proved sensitive biomarkers too, which could be used in early detection of contamination.

Experimental model studies of pesticide exposure.

The neurotoxic effects of Dimethoate (Dim), Dichlorvos (DDVP) and Methyl-Parathion (MP) respectively were investigated on the central nervous system (CNS) and peripheral nervous system (PNS) of rats after different treatment schedules at the macro and single unit cell level. At the macro investigations 1/25, 1/50 and 1/100 of the respective LD50 values of each pesticide were administered to different groups by gavage daily in the following programs: Pregnancy variation (P) to females from 5th to 15th days of pregnancy; Pregnancy and lactation variation (P+L): to females as above and during lactation for 4 weeks; Pregnancy+lactation+post weaning variation (P+L+P) as above plus to the young male rats (F1 generation) up to 8 weeks. Neurotoxicological investigations were conducted on the F1 rats at the age of 12 weeks. Spontaneous electrocorticograms (ECoG) were recorded on the anesthetized rats from the somatosensory, visual and auditory cortex. Cortical evoked potentials (EP) were recorded from the same areas subsequently. Conduction velocity and refractory periods of the tail nerve was investigated. Treatment by Dim, DDVP and MP during P and P+L of the mothers did not influence the bioelectric activity of the offsprings significantly. The same treatment by the P+L+P programme, resulted in significant changes. Frequency of the spontaneous ECoG waves grew significantly in all dose groups of P+L+P group. Latency time become shorter after somatosensory. light or acoustic stimuli respectively on one hand and the beginning of the of answer of these by the evoked potential (EP) waves on the other hand. Conduction velocity of the tail nerve diminished, refractory periods grew dose dependently and significantly at the P+L+P programs with all the three pesticides. Cortical single unit activity was studied after the i.p administration of 1/5 LD50 of the three organophosphates (OP). The decrease of the firing frequencies was observed. The amplitude of the hippocampal population spikes increased. The changes observed in these studies point toward a higher excitation state of the CNS and a disturbed conduction of the nervous impulses of the peripheral nerves. These results could be taken into consideration when deciding on human contaminations by OP-s.

Developmental neurotoxicological effects of lead and dimethoate in animal experiments.

Neurophysiological changes caused by parallel treatment with inorganic lead and dimethoate (a combination of possible health risk at population level) were investigated in different phases of the ontogenesis. Wistar rats were treated by gavage with lead (80.0 or 320.0 mg/kg); with dimethoate (7.0 or 28.0 mg/kg), or with their combination on days 5-15 of pregnancy, days 5-15 of pregnancy + days 2-28 of lactation (females of P generation), or days 5-15 of pregnancy + days 2-28 of lactation (females of P generation) + 8 weeks after weaning (males of F1 generation). Electrophysiological parameters (electrocorticogram, cortical evoked potentials) of the F1 male rats in the above groups were investigated at the age of 12 weeks. Both spontaneous and evoked electrophysiological phenomena showed dose-, treatment- and combination-dependent changes (e.g. significantly decreased mean amplitude and increased frequency of the electrocorticogram, lengthened latency and duration of the somatosensory, visual and auditory evoked potentials) which seemed to be more pronounced in the groups treated with the combination of lead and dimethoate than in the groups given lead or dimethoate alone. The results indicate that a simultaneous, pre- and postnatal exposure to the neurotoxicants, lead and dimethoate, considerably altered the functioning of the central nervous system.

Extension of the protocol of OECD guideline 407 (28-day repeated dose oral toxicity test in the rat) to detect potential immunotoxicity of chemicals.

To indicate the immunotoxic potential of chemicals the examinations prescribed by OECD Guideline 407 were extended by the following additional toxicological, haematological, histopathological, and immune function examinations: absolute and relative organ weight of spleen, thymus, popliteal lymph nodes, lung and brain; histopathology of thymus, mesenteric lymph nodes, popliteal lymph nodes, bone marrow (femur), Peyer's patches (ileum), lungs and colon; PFC assay (spleen), T cell proliferation and NK cell assay. Two well known immunosuppressants Azathioprine (AZA) and Cyclosporine A (CysA) were chosen as model compounds at a dose range which do not cause visible toxic signs on the animals during a 28 days treatment period. The results show that the applied experimental system is much more sensitive in detection of the immunotoxic potential of these two compounds in a low dose range than the examination required by OECD Guideline 407 are.