B. V. LIPPS¹

1 Ophidia Products Inc. 11320 South Post Oak, Suite 203, Houston, Texas 77035 USA.

ABSTRACT: The pharmacological substances adenosine deaminase (ADA), histamine, and IgE are endogenously present in animals. They are implicated in allergy and asthma and detectable in blood serum. This research reports the presence of ADA, histamine, and IgE at varying levels in almost all major organs of mice. This research further reports that intramuscular injection of sub-lethal dose of Naja kaouthia venom disrupted homeostasis and lowered the levels of ADA, histamine, and IgE in the organs of mice. Adult Balb/C male mice were injected with a half lethal dose of the venom. The mice were sacrificed at 2, 8, and 24 hours post-injection and different organs were collected. Organs were homogenized, centrifuged, and the supernatants were assayed for ADA, histamine, and IgE using respective antisera by immunological test enzyme-linked immunosorbent assay (ELISA). Organs from mice injected with PBS served as controls. No major decrease in the levels of ADA, histamine, and IgE was observed after 2 hours of venom injection. However, tremendous decreases in the levels of ADA, histamine, and IgE was observed in organs 24 h post-injection. The highest decrease for ADA was observed in the brain, liver, lung, muscle, and testis; for histamine, in the heart, muscle, lung, and testis; and for IgE, in the bone, heart, lung, muscle, and testis. This is a first-hand investigation showing the effect of envenomation on the pharmacokinetics of ADA, histamine, and IgE in organs.
KEY WORDS: cobra venom, Balb/C mice organs, adenosine deaminase, histamine, IgE, Naja kaouthia.

Snake envenomation is complex involving the direct action of venom components on the tissue and release of various endogenous mediators. Snake venoms are known to cause different metabolic disorders by altering the cellular and enzymatic activities in animals. Liberation of pharmacological substances by snake venoms is known (20). There are reports showing increments in cytokine levels in mice and patients bitten by Bothrops asper and Bothrops jararaca (1,13) and in sera of mice injected with Bothrops atrox venom (2). Increments in serum cytokine and nitric oxide were shown in mice injected with B. asper and B. jararaca venoms (15). Both venoms induced prominent elevations of tumor necrosis factors and interleukins IL-1, IL-6, IL-10 in injected mouse sera.

The research (16,17) reported that rabbit livers and kidneys were affected by venom injection. These investigators showed the effects of IM injection of a sub-lethal dose of the Egyptian cobra venom on the histological and histochemical pattern of rabbit kidney and liver. They concluded that nephrotoxicity should be considered as one of the serious consequences of cobra venom.

Adenosine deaminase (ADA) is an essential enzyme that controls levels of biological active purines and 2'-desoxy adenosine in tissues and cells (6). ADA deficiency results in combined immunodeficiency by a severe T, B, and NK cell lymphopenia (7). ADA deficiency is also associated with bony and renal abnormalities (19), hepatocellular damage, neurological disorders, and pulmonary insufficiency (3,9). Some ADA-deficient patients have been reported to elevate IgE and eosinophilia, which are implicated in allergy-asthma (5).

It was demonstrated that monoclonal antibodies to IgG4 induce histamine release from human basophiles in vitro (4). Evidence of histamine release was demonstrated as a principal pharmacological component of Australian wolf spider venom (18). Histamine release in humans has been reported as a consequence of honeybee and yellow jacket venom allergy (10).

The earliest demonstration of IgE antibodies against venoms in sera of victims of fatal anaphylaxis from stings has been reported (8). It was concluded that a small but appreciable portion of population has venom-specific antibodies and the prevalence is seasonally variable (23). It was reported that both IgG and IgE antibody levels rose with immunotherapy with bee and wasp venoms (10).

Published literature reveals that envenomation caused pathological and histological changes in animal organs, such as kidney and liver. There are reports showing the increased levels of histamine, IgE, and cytokines in blood sera of animals injected with venom. Interleukin-6 was released following scorpion sting in children (22). This investigation reports the endogenous presence of ADA, histamine, and IgE in organs of Balb/C mice. The organs tested were bone, brain, heart, kidney, liver, lung, muscle, pancreas, salivary gland, spleen, and testis. The investigation further reports the effect of cobra venom injection on the organs of mice, which was expressed as a loss of important pharmacological substances ADA, histamine, and IgE.

MATERIALS AND METHODS

Venom was collected from several adult Naja kaouthia snakes at various times during a calendar year and frozen. The stored venom was thawed and pooled. The pooled venom was aliquoted and stored at -20°C until used.

Production of polyclonal antibodies to IgE in rabbits

IgE was purchased from Scripps Laboratory. Anti-IgE was produced by immunizing adult New Zealand rabbits by IM of 250 mg of IgE per rabbit three times two weeks apart. The first injection consisted of a mixture of IgE and Freund's complete adjuvant (FCA). The subsequent injections consisted of IgE mixed with Freund's incomplete adjuvant (FIA).

Production of polyclonal antibodies to ADA in mice

These antibodies were produced as described by Lipps (11). ADA was purchased from Sigma-Aldrich Company. Adult Balb/C mice were immunized with ADA by IM inoculation. First injection consisted of 0.2 ml/mouse containing 100 mg of ADA in PBS mixed with equal volume of FCA. Subsequently, three additional injections were given two weeks apart, consisting of ADA at the same concentration mixed with FIA. Immunized mice were bled two weeks after the last injection through the ophthalmic vein and serum was collected.

Inoculation of mice with sub-lethal dose of venom

The lethal dose for N. kaouthia venom was determined in mice by IM injection at various concentrations. The lethal dose for mouse was found to be 4 mg. Therefore, mice were injected with 2 mg in 0.2 ml. Twelve mice were used, 3 injected with 0.2 ml PBS as control and 9 with venom.

Preparation of organ suspension

Initially, two normal male mice were sacrificed for their organs, and the suspensions were tested for ADA, histamine, and IgE by ELISA. The organs tested were bone, brain, heart, kidneys, liver, lung, muscle, pancreas, salivary glands, skin, spleen, and testis. The detectable presence of ADA, histamine, and IgE in mouse organs initiated these studies into the effect of venom injection on the organs with respect to these endogenous pharmacological substances.

Mice injected with venom were sacrificed after 2, 8, and 24 hours. The control mice were sacrificed along with 8 hours venom injected mice. Organs from three mice for each category were collected and pooled. The pool of organs was homogenized in PBS using a manual homogenizer. Bone and skin were crushed by mortar and pestle before homogenizing. The homogenates were centrifuged and the supernatants for each pool organ were separated. Protein concentration for each supernatant was measured by spectrophotometer using a protein kit from Bio-Rad (catalogue 500-0006). Supernatant protein concentrations were adjusted to 100 mg/ml with PBS and kept as stocks for further testing.

ELISA tests were performed in 96 well microtiter plates (12). The reagents for ELISA were purchased from Sigma-Aldrich Co. The stocks from mice organs were diluted with carbonate-bicarbonate buffer, pH 9.4, to 10 mg/ml as coating antigen for ELISA. The wells of the plate were coated with antigen, 100 ml/well. The plate was left at room temperature (RT) overnight, after which it was emptied and washed three times (3x) with PBS, and the wells were blocked with 250 ml/well of 3% fish skin gelatin. After 30 min, the plate was emptied and washed 3x with PBS. Antisera against ADA, histamine, and IgE diluted in gelatin were added. Three wells received 100 ml of each dilution of antiserum, diluted two-fold from 1:300. The antigen controls without antibody were incorporated. The plate was incubated at 37°C in a humid incubator for 1 h after which the plate was washed 3x with PBS. The horseradish peroxidase conjugated with mouse IgG for ADA, histamine, and IgE assay, and the horseradish peroxidase conjugated with rabbit IgG 100 ml/well were reacted for 30 min. Finally, after washing, the wells were reacted with O-phenylnediamine-HCl for color development. The plate was read after 30 min, and the OD was recorded at 405 mm as ELISA titer per 100 ml.

RESULTS

The results showed that ADA, histamine, and IgE were present in all tested organs including bone and skin at varying degree level. ADA levels in muscle were the highest followed by the brain, liver lung, and testis. Histamine level was higher in lung than the other organs. IgE level was found to be the highest in testis followed by the bone, heart, lung and muscle.

Table 1. ADA, histamine, and IgE concentrations in organs of adult male mice expressed as ELISA titer/100 µl.

Figure 1. ADA levels in organs of mice injected with N. kaouthia venom. Balb/C mice were injected with venom, and the organs were collected after 2, 8, and 24 hours, homogenized, and tested for ADA by ELISA.

Figure 2. Histamine levels in organs of mice injected with N. kaouthia venom. Balb/C mice were injected with venom, and the organs were collected after 2, 8, and 24 hours, homogenized, and tested for histamine by ELISA.

Figure 3. IgE levels in organs of mice injected with N. kaouthia venom. Balb/C mice were injected with venom, and the organs were collected after 2, 8, and 24 hours, homogenized, and tested for IgE by ELISA.

Investigations on the changes taking place in blood serum as a consequence of envenomation in humans and experimental animals is a subject of many years of research. Increased glucose and lactate concentrations were known to be common during snake envenomation (14). Increased levels of various cytokines have been reported as a consequence of snake and scorpion envenomation (22).

ELISA assay was developed for venom detection in autopsy specimens of mice and human victims (21). These investigators found the lowest concentrations of venom in brain with moderate amounts in liver, spleen, kidney, and lungs. Studies of histological and histochemical alterations in liver and kidneys of rabbits injected with the sub-lethal dose of Egyptian cobra venom are reported (17).

Thus, the aftermath of envenomation in experimental animals and human snakebite victims has been investigated by a variety of methods, using blood serum and only the kidneys and livers. Increments in the levels of various cytokines in serum and pathological changes occurring in the liver and kidney have been reported (17). ADA, histamine, and IgE are important pharmacological substances, which are endogenously present and implicated in allergy-asthma. This research showed that these pharmacological substances are present in all major organs of mice. The results showed that snake envenomation caused stress in mice, disrupting homeostasis and affecting the organs to lose ADA, histamine, and IgE.

The levels of endogenously substances ADA, histamine, and IgE in organs of mice were assayed by ELISA using respective antisera. In these studies, ADA, histamine, and IgE were found to be present at different levels in all tested organs. Sub-lethal IM injection of cobra venom in male Balb/C mice decreased the levels of these substances in all organs. The level of cytokines increased in blood sera after envenomation (16). Assuming that what is lost from the organs must get into the circulation, it follows that ADA, histamine, and IgE should show increased levels in blood sera of mice but will not reveal which organs were affected. In order to assay the increased levels of ADA, histamine, and IgE in mouse serum, sandwich-type ELISA is required, which cannot be compared with the simple ELISA results. The thrust of this research is to provide a new method to show the effect of cobra envenomation on the organs.

The results of our research show decreased levels in various mice organs at 24 hours. Future research should be directed to: 1- determine the period for decreased levels of ADA, histamine, and IgE in organs of mice injected with cobra venom; the recovery period of these substances to normal levels should be determined by mice organ assays at 72 hours, 1, and 2 weeks post-injection along with appropriate controls; and 2- to determine the prevention of ADA, histamine, and IgE loss in mice organs injected with cobra venom if treated with anti-cobra venom, or natural and synthetic lethal toxin neutralizing factors (12) within 2 hours.

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Received Jannuary 2, 2001
Accepted March 1, 2001

CORRESPONDENCE TO:
B. V. LIPPS. Ophidia Products, Inc. 11320 South Post Oak, Suite 203, Houston, Texas 77035, USA. Telephone: 713-723-6845 and Fax: 713-663-7290.
E-mail: bvl@ophidia.com