Liver hypertrophy: a review of adaptive (adverse and non-adverse) changes--conclusions from the 3rd International ESTP Expert Workshop.

Preclinical toxicity studies have demonstrated that exposure of laboratory animals to liver enzyme inducers during preclinical safety assessment results in a signature of toxicological changes characterized by an increase in liver weight, hepatocellular hypertrophy, cell proliferation, and, frequently in long-term (life-time) studies, hepatocarcinogenesis. Recent advances over the last decade have revealed that for many xenobiotics, these changes may be induced through a common mechanism of action involving activation of the nuclear hormone receptors CAR, PXR, or PPARα. The generation of genetically engineered mice that express altered versions of these nuclear hormone receptors, together with other avenues of investigation, have now demonstrated that sensitivity to many of these effects is rodent-specific. These data are consistent with the available epidemiological and empirical human evidence and lend support to the scientific opinion that these changes have little relevance to man. The ESTP therefore convened an international panel of experts to debate the evidence in order to more clearly define for toxicologic pathologists what is considered adverse in the context of hepatocellular hypertrophy. The results of this workshop concluded that hepatomegaly as a consequence of hepatocellular hypertrophy without histologic or clinical pathology alterations indicative of liver toxicity was considered an adaptive and a non-adverse reaction. This conclusion should normally be reached by an integrative weight of evidence approach.

Serologic comparisons of 4 Anaplasma isolates as measured by the complement-fixation test.

Anaplasma marginale isolated from Virginia ( VAM ), from North Texas ( NTAM ), from Florida (FAM), and Anaplasma ovis from Idaho were used in these trials. Complement-fixation antigens from each of the 4 isolates were used to compare complement-fixing antibody titers of 10 cattle infected with VAM , 17 with FAM, and 6 with NTAM . Strong cross-reactions occurred with all antigens and sera. The homologous system generally showed higher average antibody titers. The serum antibody titers occurring with the A. ovis antigen were significantly lower than those seen with A. marginale antigens. Serum antibody titer differences as measured by FAM and NTAM antigens on sera from VAM , NTAM and FAM failed to reach significance. Serum antibody titer comparisons using VAM antigen were significantly different from those occurring with FAM and NTAM in most instances.

Long-term inhalation toxicity of N-vinylpyrrolidone-2 vapours. Studies in rats.

In previous subchronic studies inhaled N-vinylpyrrolidone-2 (NVP) was haemotoxic, hepatotoxic and irritant to the nose. In the first of two long-term studies, study A, Sprague-Dawley rats were exposed by inhalation to 0, 5, 10 or 20 ppm NVP (6 hr/day, 5 days/wk) for 24 months. Satellite groups were killed after 3, 12 or 24 months. In study B, female Sprague-Dawley rats were exposed to 0 or 45 ppm NVP for 3 months and killed at 3 or 12 and 24 months post-exposure. In study A, survival was unaffected, but reduced body weight gain, haemotoxicity, effects on clinical chemistry parameters indicative of hepatotoxicity, increased liver weight, hepatocellular carcinomas, necrosis, reparative hyperplasia, adenomas and adenocarcinomas of the nasal cavity, and squamous cell carcinomas of the larynx were seen. Increased tumour incidence was seen only in the liver and upper respiratory tract. In study B, the effect of NVP on body weight evident at 3 months disappeared before 1 yr, but effects on liver pathology persisted throughout the subsequent 21-month exposure-free period, and a few liver tumours were seen at 2 yr. As NVP gave negative results in a battery of in vitro and in vivo genotoxicity tests, it appears that the tumours that arose were manifestations of a non-genotoxic mechanism.

Subchronic inhalation and oral toxicity of N-vinylpyrrolidone-2. Studies in rodents.

N-Vinylpyrrolidone-2 (NVP) is a monomeric compound used as an industrial intermediate. Nine of 11 studies previously reported involved exposure of rats (two different strains), mice or hamsters to NVP by the inhalation route at concentrations of up to 120 ppm (6 hr/day, 5 days/wk) over a period of 1 wk to 12 months. The remaining two studies involved exposure of rats to NVP through the drinking water or by gavage at dose levels of up to 100 mg/kg body weight/day. Reduced body weight gain was seen in rats exposed by inhalation to 5 ppm or more for 3 months and in mice and hamsters exposed to 45 ppm for only 1 day. Effects were seen on haematological (reduced haemoglobin, erythrocyte count, haematocrit) and clinical chemistry parameters (specially raised gamma-glutamyltransferase activity and decreases in plasma protein), liver weight increase and liver lesions (centrilobular single-cell necrosis and foci of hepatocellular alteration) in rats and mice but not hamsters. Rats exposed to 40 mg/kg body weight/day NVP or more for 3 months by gavage developed similar liver changes. Atrophy of olfactory epithelium and hyperplasia of nasal respiratory epithelium was seen in rats exposed by inhalation to 5 ppm NVP for 7 wk but not in response to 1 ppm for 13 wk (no observed-adverse-effect level, NOAEL). These studies indicated that the upper respiratory tract and the liver are the main targets for NVP toxicity.

Chemoprophylactic activity of imidocarb, diminazene and oxytetracycline against Babesia bovis and B. bigemina.

Splenectomized calves treated with imidocarb, diminazene, and oxytetracycline were exposed to Babesia bigemina and B. bovis stabilates at various time intervals following treatment to evaluate prophylactic efficacy. Diminazene showed no residual activity against a B. bigemina challenge given 54 days after treatment. Oxytetracycline appeared responsible for an increased incubation time when given 2 days prior to B. bigemina exposure. Imidocarb showed marked prophylactic efficacy against both B. bigemina and B. bovis. Treatment with 1 or 2 mg kg-1 imidocarb, followed by Babesia exposure on the day of treatment, 7 days after treatment, then every 14 days for 91 days, delayed patent B. bigemina infections for 49 days and patent B. bovis infections for 42 days. Imidocarb at 4 or 5 mg kg-1, followed by similar Babesia exposures, delayed patent B. bovis infections for 68 days, and delayed B. bigemina for 81-103 days, and in some instances prevented infections. The delayed onset of infection due to either B. bigemina or B. bovis, following imidocarb treatment was accompanied by a significantly milder clinical response. Calves not responding to the primary challenge were fully susceptible to stabilate challenge 196 days after treatment. Calves experiencing a mild clinical response to B. bovis following imidocarb treatment and exposure failed to show any signs of response to a 196-day challenge exposure. Calves experiencing a mild clinical response to B. bigemina following imidocarb treatment and exposure did, in some instances, show a second mild response when challenged 196 days after initial treatment.

Attenuation of Babesia bovis by in vitro cultivation.

A virulent strain of Babesia bovis was adapted to grow in erythrocyte culture in the presence of equine serum and in lieu of bovine serum. Four splenectomized calves inoculated with the adapted strain, 429, developed hematologic signs of infection and a low grade fever, but remained free of central nervous system (CNS) signs and recovered. All of six control animals inoculated with a virulent strain reacted severely and five showed CNS signs and died. The calves injected with the attenuated strain were solidly immune when challenged with the virulent strain at 44 or 78 days after vaccination.

The pathogenicity and immunologic relationship of a virulent and a tissue-culture-adapted Babesia bovis.

Babesia bovis grown in tissue culture was used to inoculate 12, 2-year-old Holstein steers. All 12 developed serological evidence of infection but only six had a febrile response of greater than or equal to 40 degrees C, and only one had a demonstrable B. bovis parasitemia. An average modest drop of 19% was observed in packed cell volume (PCV) during the period of reaction. All 12 steers were subsequently challenged with virulent B. bovis: seven on day 78 post inoculation (p.i.), two on day 106 p.i., and three on day 251 p.i. No demonstrable clinical response was observed in any of the 12 steers previously exposed to the tissue-culture organism, whereas severe signs of babesiosis occurred in seven 2-year-old, non-vaccinated control steers given a comparably virulent B. bovis challenge. All seven controls showed a febrile response, B. bovis parasitemias, with an average drop of 55% in PCV and a 28% mortality.

Evaluation of a cloned Babesia bovis organism as a live immunogen.

A Babesia bovis isolate was cloned by in vitro cultivation and compared to the original cultured isolate for pathogenicity by animal inoculation. Four yearling heifers were inoculated with cloned material and 4 with the original culture. The four animals which received the cloned Babesia showed comparatively minor hematologic changes and no clinical signs. One animal died in the group that received uncloned Babesia and the mean temperature increase and mean reduction in packed cell volume (PCV) was greater in that group. The four animals receiving the cloned material were challenged 100 days following initial inoculation. All of the animals were totally immune with no significant change in temperature or decrease in PCV, whereas control (previously non-inoculated) animals developed significant (P less than 0.001) increases in temperature and severe anemia. The cloned organism appears to be a candidate live immunogen for use in endemic areas to induce protection against bovine babesiosis.

Observations on anaplasmosis following field exposure to heavy and light infestations with Boophilus microplus.

Two groups of Normandy calves were exposed to heavy (mean half body count = greater than 300) or light (mean half body count = 9) natural infestations with Boophilus microplus. All of the calves became infected with Anaplasma marginale. Despite the difference in tick challenge level, there was no significant difference in the incubation period, increase in body temperature, level or duration of Anaplasma parasitemia, decrease in packed cell volume, or complement-fixing antibody response, between the heavily and lightly infected calves. Neither the incubation period nor the clinical severity of anaplasmosis was significantly influenced by the number of infested tick vectors.

Viability and virulence of Babesia rodhaini eight years after cryogenic preservation with dimethyl sulfoxide.

Mouse blood infected with Babesia rodhaini and containing an equal volume of 4 M dimethyl sulfoxide was infective after storage at -196 degrees C for 8 years. The Babesia organisms were still able to cause lethal infections after prolonged low temperature storage.

Serologic response of Babesia equi-infected horses as measured by complement-fixation and indirect fluorescent antibody tests.

Both the complement-fixation test (CFT) and the indirect fluorescent antibody test (IFAT) were conducted on weekly serum samples from nine Arab geldings for 28 days before and 256 days after their exposure to Babesia equi of European origin. On an average the IFAT became positive 8 days before the CFT and showed higher relative serum titer increases. Both test procedures successfully detected infection and neither showed an appreciable drop in titer during this time frame, with the exception of the CFT, which showed a transient drop immediately following treatment with imidocarb. A test conducted 540 days after infection showed four of the eight surviving, and presumably infected, horses to be negative on CFT, where as all eight were still positive on IFAT. Comparisons made with the IFAT, on horse sera from B. equi infection of both European and North American origin, utilizing homologous and heterologous antigens, showed significantly higher titers with homologous antigens.

The effect of Imidocarb treatment on Babesia in the bovine and the tick (Boophilus microplus).

Treatment of calves with 5 mg/kg Imidocarb (3,3-1-bis-(2 - imidazolin - 2 - yl)carbanilide dipropionate) given intramuscularly 14 days before and 14 days after exposure to Babesia infected Boophilus microplus larvae rendered the next generation of larvae incapable of transmitting Babesia infection. When administered to calves 14 or 28 days before tick exposure, the drug prevented the development of clinical babesiosis; the larval progeny of ticks reared on the calf which was treated 28 days before infestation were infective. Treatment of a calf 42 days before exposure to infective larvae did not prevent the development of a Babesia parasitaemia but appeared to reduce the severity of infection.

Subchronic toxicity studies of 3-methyl-1-butanol and 2-methyl-1-propanol in rats.

1. 90-day subchronic toxicity studies with 3-methyl-1-butanol (MEB) and 2-methyl-1-propanol (MEP) were performed on rats to evaluate the toxicological profile of the compounds under conditions of drinking water studies, to identify the potential target organs, and to determine no-observable-adverse-effect-levels (NOAELs) respective of the substances. The test substances were administered to groups of 10 male and 10 female Wistar rats in drinking water at concentrations of 0, 1000 p.p.m. (about 80 mg/kg/d), 4000 p.p.m. (about 340 mg/kg/d) and 16,000 p.p.m. (about 1250 and 1450 mg/kg/d of MEB and MEP respectively). 2. 16,000 p.p.m. was found to be the maximal concentration for both alcohols applicable to rats in drinking water. Higher concentrations had an influence on palatability and could thus not be tested in drinking water studies. 3. At 16,000 p.p.m. MEB a marginal increase in the red blood cell count as well as a slight decrease in the mean corpuscular volume and the mean corpuscular hemoglobin content was observed in males only. These changes are considered to be treatment-related, although the toxicological significance of these findings is unclear. No other substance-related effects were found on body weight (b.w.), mortality, various parameters of clinical chemistry, organ weights, gross pathology and histopathology. 4000 p.p.m. MEB did not cause any substance-induced changes. Therefore, the NOAEL of MEB was defined as 4000 p.p.m. for male and 16,000 p.p.m. for female rats under conditions of oral application via drinking water. 4. MEP concentrations up to and including 16,000 p.p.m. did not induce any signs of toxicity and were therefore defined as the NOAEL respective of this substance for rats under conditions of drinking water application.

Influence of a second Anaplasma exposure on the success of treatment to eliminate Anaplasma carrier infections in cattle.

Treatment of adult Anaplasma carrier cows, with long-acting oxytetracycline at dosage levels generally successful in eliminating infection, was unsuccessful when the treatment was preceded or accompanied by a 2nd exposure to A marginale on days 0, 7, or 14 before treatment. Noninfected calves exposed to A marginale 7 days before a similar treatment developed anaplasmosis and became carriers of infection.

Infection of splenectomized calves with Anaplasma ovis.

An Anaplasma was not recovered from 2 splenectomized calves at 17 days after A ovis inoculation, but was recovered from 1 of the calves at 177 days after inoculation. In a 3rd calf exposed to A ovis, an Anaplasma was recovered at 177 and 262 days after inoculation. the Anaplasma recovered from these calves was determined to be A ovis on the basis of cross-infectivity trials, using Anaplasma marginale.

Anaplasma marginale in bovine erythrocyte cultures.

Bovine erythrocytes infected with Anaplasma marginale were standardized with normal bovine erythrocytes at 1.4% and 3.5% parasitemia and cultured under low oxygen pressure at 37 C. The number of erythrocytes infected by A marginale appeared to increase 3.3-fold by day 11 and 4.5-fold by day 3, as determined by microscopic examination of Giemsa-stained thin films of the culture suspension. Anaplasma parasites in experimental cultures were proven to be viable and pathogenic by calf inoculation for up to 16 days.

Immunization of cattle with a Babesia bigemina antigen in Freund's complete adjuvant.

Three adult intact cattle and five splenetomized calves were immunized against Babesia bigemina by the subcutaneous injection of an adjuvant-vaccine given twice at 3- and 4-week intervals, respectively. The antigen consisted of B bigemina parasites and erythrocytic stroma. The vaccinated adults plus one control, and the vaccinated splenectomized calves plus four comparable controls were challenge exposed at 67 and 33 days, respectively, after the last vaccine injection. All control animals died of acute babesiosis. The intact vaccinated adult cows showed only minimal response and little or no drop in packed cell volume. Three of five vaccinated splenectomized calves showed a moderate response to challenge exposure and recovered rapidly.

Isolation of a bovine Theileria.

Dual infections of Anaplasma marginale and a Theileria, resembling Theileria mutans, occurred in splenectomized calves inoculated with pooled blood samples from eastern Texas cattle. Theileria was obtained in pure form by treating dually infected cattle with selectively eliminated Anaplasma. These theileria infections were responsible for mild, transient reductions in packed red blood cell volume (PCV).

Bovine anaplasmosis: in utero transmission and the immunologic significance of ingested colostral antibodies.

Neonate progeny from 3 Anaplasma-free (clean) and 5 Anaplasma-carrier cows were splenectomized and each was challenge exposed with 5 ml of carrier blood. Prepatent times were between 18 and 25 days in calves born of clean cows and between 21 and 36 days in progeny from carrier dams. The lowest packed cell volume values in the clean group occurred at 25 to 39 days after the challenge inoculation and at 29 to 47 days in the carrier group. Highest parasitemias in the clean-calves ranged between 13% and 51% in 25 to 35 days and between 35% to 64% in 38 to 43 days in the carrier calves. Seven splenectomized calves were inoculated with 60 ml of whole blood from progeny of known Anaplasma-free or Anaplasma-carrier cows. After 183 days, all but 1 calf remained free of anaplasmosis. A 1% parasitemia was first observed in that calf 12 days after inoculation with blood from a calf which showed signs of acute anaplasmosis at birth. The infected neonate's dam had recovered from acute anaplasmosis infection during the middle of the second trimester of the gestation. Although not statistically significant, colostral antibodies and/or other maternal factors did not seem to completely protect progeny, but lengthened the prepatent period and delayed anemia and the climax of parasitemia. Further, it was determined that it was possible for an animal affected with acute anaplasmosis before the 190th day of the gestation to transmit anaplasmosis in utero.

Influence of dexamethasone on the recrudescence of Anaplasma marginale in splenectomized calves.

Dexamethasone was administered at the dose rate of 0.2 mg/kg of body weight to 11 splenectomized Anaplasma-carrier calves (groups 1 and 3) on Monday, Wednesday, and Friday for 3 weeks. Observations were made on these calves and on 7 nontreated, comparable calves (group 2) to determine the influence of treatment on carrier infections. Dexamethasone treatment was associated in every instance with an exacerbation of the Anaplasma parasitemia and a decrease in packed red cell volume. The episode of acute anaplasmosis was of short duration, resembling the primary response, except that complement-fixation response did not increase accordingly. Serum protein electrophoresis of serums from 4 calves (group 3) undergoing the drug-induced response failed to show any significant change during the 3-week treatment period, but did show a significant increase in gamma-globulin immediately after treatment.