Application of Quantitative Analysis of Diatoms in Lung Tissue for the Diagnosis of Drowning of Experimental Animals.

ABSTRACT: Objective To discuss the application value of diatom examination in lung tissue for the forensic diagnosis of drowning. Methods The experimental animals were divided randomly into drowning, postmortem submergence and dying on land group. Diatoms in lung tissue and drowning fluid were analyzed quantitatively by microwave digestion-vacuum filtration-automated scanning electron microscopy diatom examination method. The ratios of content of diatoms in lung tissue and drowning fluid （CL/CD ratio） were recorded. Results The CL/CD ratios of experimental rabbits in the drowning group （5.82±3.50） were much higher than that of postmortem submergence group （0.47±0.35）; the CL/CD ratios of different parts of the lung lobes of experimental pigs in the drowning group were higher than that of postmortem submergence group （P<0.05）; in seawater, brackish water, river fresh water and lake fresh water, the CL/CD ratios of experimental pigs in the drowning group were higher than that of postmortem submergence group （P<0.05）. In animal experiments, all the cases with CL/CD ratio >1.6 were from drowning group. Conclusion CL/CD ratio is an indicator with good application prospects in the diagnosis of drowning.

Electron Microscopic Study in the Rat Model of Electrically Injured Myelopathy: Preliminary Report.

Objective: The patient with electrically injured myelopathy showed mild motor weakness without somatosensory pathway abnormalities. Few reports have been reported on the pathophysiological mechanisms of electrically injured myelopathy, and there is controversy about the exact pathological causes. This study aimed to investigate the ultrastructural changes in the electron microscopic findings of electrical spinal cord injury. Methods: Nine rats were used in this study. We performed 7 electrical shocks (frequency, 120 Hz; pulse width, 0.9 ms; duration, 3 seconds; current, 99 mA) using an electroconvulsive therapy (ECT) apparatus (57800 ECT unit; UGO BASILE). We used one ear and one contralateral hind limb as entry and exit sites, respectively. We only enrolled rats with hind limb weakness and performed electron microscopy evaluations of the spinal cord on the first day and 4 weeks after injury. Results: On the first day after injury, an electron microscopic examination showed a directly damaged area that appeared to be torn as physical damage, damaged myelin sheath, vacuolated axons in the myelin sheath, swollen Golgi apparatus, and injured mitochondria. Looking at changes in motor and sensory nerves, the sensory neurons showed recovered mitochondria and Golgi apparatus 4 weeks after injury; however, motor neurons still showed injured mitochondria, swollen Golgi apparatus, and endoplasmic reticulum. Conclusion: This study showed that recovery from ultrastructural injury was more rapid in sensory neurons than in motor neurons.

Immunological effects of thermal injury. I. Inhibition of spermatogenesis in guinea pigs.

PIP: In the present study, controlled burns of 1 testis have induced the formation of testis-specific antibodies in Hartley guinea pigs. The antibodies reacted with autologous as well as homologous testicular extracts. In addition, pathological changes have been noted in the germinal components of the contralateral testis, which were similar to those observed after the induction of experimental allergic orchitis by active immunization with testicular tissue. Results of the study indicate that thermal injury to guinea pig testes can induce an autoimmune response similar to that observed after immunization with autologous or homologous testicular tissue. The antibodies formed were organ-and species-specific against a testicular antigen. Thermal injury may be associated with autoimmunization of the host by the injured organ.^ieng

Developmental changes in mitochondria during the transition into lactation in the mouse mammary gland.

Mitochondrial biogenesis in the parenchymal cell of the mouse mammary gland appears to occur in two distinct phases: replication during cell proliferation, and maturation during cell differentiation. This study of the mitochondrial maturation phase in the mouse gland demonstrates a significant increase in organelle density on isopycnic sucrose gradient centrifugation during the transition from late pregnancy to day 8 of lactation. Differential fragility to high sucrose concentrations or changes in mitochondrial lipid composition do not satisfactorily explain the density increases. When organelle densities were assessed by centrifugation under iso-osmotic conditions with Ficoll gradients in 0.25 M sucrose, the mitochondria from pregnant glands were observed to be more dense than those from lactating glands. The two mitochondrial populations were also found to differ in their response to changes in sucrose concentration in the Ficoll gradients. When sucrose concentration was increased, the density of both pregnant and lactating gland mitochondria increased nonlinearly, the increase being greater with the lactating gland organelles. By use of mathematical models, the differing response was interpreted as a change in the density and osmotic activity of the mitochondrial internal compartment (inner membrane plus matrix space). We have proposed that the changes reflect a large expansion of the inner mitochondrial membrane and perhaps the matrix material during the transition into lactation in the differentiating parenchymal cell.

PIP: Developmental changes in mitochondria during pregnancy and the transition into lactation in the parenchymal cell of the mouse mammary gland were studied. There were 2 apparent distinct phases in mitochondrial biogenesis: replication during cell proliferation and maturation during cell differentiation. Isopycnic sucrose gradient centrifugation during the transition from late pregnancy to Day 8 of lactation revealed a marked increase in organelle density. This increase in organelle density could not be explained by differential fragility to high sucrose concentrations or changes in mitochondrial lipid composition. Mitochondria from pregnant glands were more dense than those from lactating glands as determined by centrifugation under iso-osmotic conditions with Ficoll gradients in .25 M sucrose. These mitochondrial populations also differed in their response to changes in the concentration of sucrose in the Ficoll gradients. The density of both pregnant and lactating gland mitochondria increased nonlinearly when the sucrose concentration was increased, with the increase in density being greater in the latter. The difference in response was mathematically interpreted to reflect a change in the density and osmotic activity of the mitochondrial internal compartment (inner membrane plus matrix space). It is proposed that the developmental changes observed reflect a large expansion of the inner mitochondrial membrane and possibly the matrix material during the transition into lactation in the differentiating parenchymal cell.

Developmental changes in mitochondria during the transition into lactation in the mouse mammary gland. II. Membrane marker enzymes and membrane ultrastructure.

The activity of cytochrome oxidase (an inner mitochondrial membrane marker) in mouse mammary gland homogenates was found to increase five- to sixfold from late pregnancy to day 8 of lactation, while that of monoamine oxidase (an outer membrane marker) increased only about 25%. The specific activity of cytochrome oxidase in the isolated mitochondria decreased slightly over the same period while the specific activity of monoamine oxidase decreased fivefold. This reflects the fact that both cytochrome oxidase and mitochondrial protein are increasing at a much greater rate than is monoamine oxidase activity. Mixing experiments preclude the possibility that the release or removal of an inhibitor or stimulator produces the changes in enzymatic activity. The cytochrome oxidase to monoamine oxidase ratio was followed throughout the pregnancy-lactation cycle in total mammary homogenates, isolated mammary parenchymal cells, and isolated mammary mitochondria. In each preparation the pattern was the same with little change in the ratio until late pregnancy; and then a three- to fourfold increase occurred and the values reached a maximum by day 8 of lactation. These experiments were interpreted as demonstrating that the observed enzymatic changes are reflective of alterations in the mitochondria of the mammary parenchymal cell population. Electron micrographs of mid-pregnant and mid-lactating mammary parenchymal cells in situ were prepared, and distinct changes in the mitochondrial morphology noted. The most significant and obvious change is the large increase in the number of inner membrane cristae and an increase in matrix density in the lactating gland cell. Therefore, both enzymatic and morphological studies support the concept of an expansion of the mitochondrial inner membrane during presecretory differentiation in the mouse mammary parenchymal cell.

PIP: The enzyme markers for mitochondrial inner and outer membranes throughout the pregnancy-lactation cycle in the mouse were compared. The ultrastructural changes of the organelle during the transitions were studied by electron microscopy. The activity of cytochrome oxidase in mouse mammary gland homogenates was found to increase 5- to 6-fold from late pregnancy to Day 8 of lactation, while that of monoamine oxidase in the isolated mitochondria decreased slightly over the same period while the specific activity of monoamine oxidase decreased 5-fold. The cytochrome oxidase to monoamine oxidase ratio was followed throughout the pregnancy-lactation cycle in total mammary homogenates, isolated mammary parenchymal cells, and isolated mammary mitochondria. The pattern was the same in each preparation with little change until late pregnancy and then a 3- to 4-fold increase occurred and values reached a maximum by Day 8 of lactation. Electron micrographs of midpregnant and midlactating mammary parenchymal cells in situ were prepared, and changes in the mitochondrial morphology noted. The most significant change is the large increase in number of inner membrane cristae and an increase in matrix density in the lactating gland cell.

The molecular biology of RU486. Is there a role for antiprogestins in the treatment of breast cancer?

PIP: Considerable animal research and clinical trials demonstrate that progesterone antagonists could treat hormone-dependent breast cancers. Since endogenous progesterone does include mitosis in epithelial cells of the breast, in theory, exogenous progesterone can cause breast cancer. Thus administration of progesterone antagonists could block endogenous progesterone. Yet we do not know the mitosis pattern in breast cancer cells during the menstrual cycle, so research obtaining such data is needed. Ethical problems arise, however, since researchers need multiple breast tumor samples to analyze proliferative activity at various times during the cycle. A possible solution is using an aspirated tumor sample for initial mitotic analysis immediately followed by RU-486 treatment then tumor removal 24 hours later for reanalysis. Ideally well controlled studies using organ-cultured human breast tumors, human breast cancer lines, and human tumors implanted into nude mice are needed to understand the mechanisms of the mitogenic actions of progestins and progestin antagonists. Progestin antagonist may be used to treat locally advanced or metastatic cancers either as an adjuvant endocrine therapy alone or with tamoxifen. An obstacle to longterm use of RU-486 as a treatment for breast cancer is its antiglucocorticoid side effects. But the molecules of newer progesterone antagonists appear to produce maximal antiprogestin activity and minimal antiglucocorticoid activity. In addition, if RU-486 is administered with drugs that prevent adrenal steroidogenesis or peripheral aromatization of adrenal steroids to estrogens, women may take it for longterm treatment. Researchers must have the opportunity to continue basic tumor biological and molecular research to gain an understanding of the exact molecular targets and mechanisms of antagonist action. The current political climate in the US hinders such research, however.^ieng

Indicadores morfométricos de la corteza cerebral frontal de gazapos de ratas Wistar con diabetes pregestacional

Introducción: La diabetes es una enfermedad que afecta el embarazo provoca complicaciones fetales; dentro de ellas son frecuentes las malformaciones congénitas. Por la imposibilidad práctica y ética de estudiar este proceso en gestantes es imprescindible realizar estudios experimentales empleando procedimientos morfométricos para determinar si la diabetes afecta el neurodesarrollo. Objetivo: Caracterizar morfométricamente la sustancia gris de gazapos de ratas Wistar normales y con diabetes mellitus pregestacional. Métodos: Se realizó un estudio experimental básico de serie de casos a 20 gazapos de ratas Wistar de los cuales 10 eran descendientes de diabetes pregestacional. Se caracterizaron indicadores morfométricos del tejido nervioso como espesor de corteza e indicadores nucleares como el perímetro. Resultados: La media de la altura de la sustancia gris cortical mostró un valor de 1,224 ± 303,7 μm para el grupo control y 1,014 ± 376,0 para los casos, al aplicar el test de diferencias de medias se encontró diferencia significativa (p ≤ 0,05) a favor del grupo control. Los valores de la media del perímetro nuclear en el grupo control fue de 42,80 ± 7,23 μm y en el grupo experimental el promedio fue de 39,68 ± 6,52 μm, al aplicar el test de diferencias de medias se encontró diferencia significativa (p ≤ 0,05) a favor del grupo control al presentar mayor perímetro nuclear. Conclusiones: El mayor espesor cortical y perímetro nuclear correspondió al grupo control evidenciándose el efecto deletéreo de la diabetes mellitus en el neurodesarrollo.

Introduction: Diabetes is a disease that affects pregnancy causing fetal complications; within them congenital malformations are frequent. Due to the practical and ethical impossibility of studying this process in pregnant women, it is essential to carry out experimental studies using morphometric procedures to determine if diabetes affects neurodevelopment. Objective: To characterize morphometrically the gray matter of kits from normal Wistar rats and those with pregestational diabetes mellitus. Methods: A basic experimental study of a series of cases was carried out on 20 young Wistar rats, of which 10 were descendants of pregestational diabetes. Morphometric indicators of the nervous tissue were characterized as thickness of the cortex and nuclear indicators such as perimeter. Results: The average height of the cortical gray matter showed a value of 1.224 ± 303.7 μm for the control group and 1.014 ± 376.0 μm for the cases. When applying the mean difference test, a significant difference was found (p ≤ 0.05) in favor of the control group. The values of the measurement of the nuclear perimeter in the control group was 42.80 ± 7.23 μm and in the experimental group the average was 39.68 ± 6.52 μm. When applying the mean different test, a significant difference was found (p ≤ 0.05) at favor of control group presenting greater nuclear perimeter. Conclusions: The greatest cortical thickness and nuclear perimeter corresponded to the control group, evidencing the deleterious effect os diabetes mellitus on neurodevelopment.

La hipertensión arterial en animales de laboratorio / High blood pressure in experimental animals

Las Ciencias Médicas y Biológicas requieren, prioritariamente, que la investigación y la experimentación sean desarrolladas sobre organismos completos (los modelos animales). Su utilización ha permitido desarrollar innumerables ensayos preclínicos para evaluar los mecanismos patógenos y terapéuticos de diversas enfermedades, así como el estudio de las causas, naturaleza y cura de múltiples desórdenes de la salud humana. En este trabajo se muestra una panorámica general de los biomodelos de hipertensión arterial donde se describen: conceptos, características, origen, importancia, utilidad y procedimientos experimentales durante su fase de inducción. También se pondera la justificación de los biomodelos empleados en los estudios preclínicos de esta enfermedad. De igual forma, se describen los antecedentes para medir las alteraciones, las técnicas y los métodos directos e indirectos de medición de la presión arterial, la cual fue provocada experimentalmente en los animales de laboratorio para realizar los estudios de hipertensión humana.

Medical and biological sciences require, as a priority, that research and experimentation be carried out on complete organisms (animal models). Its use has allowed the development of innumerable preclinical tests to evaluate pathogenic and therapeutic mechanisms of various diseases, as well as to study the causes, nature and cure of multiple human health disorders. In this work, we show a general overview of arterial hypertension biomodels where concepts, characteristics, origin, importance, utility and experimental procedures during their induction phase are described. The justification of the biomodels used in preclinical studies of this disease is also considered. Antecedents are also described to measure alterations, techniques and direct and indirect methods of measurement of arterial pressure, which was provoked experimentally in the laboratory animals to carry out the studies of human hypertension.

Synthesis of very low density lipoproteins in the cockerel. Effects of estrogen.

The effect of estrogen on the synthesis of plasma very low density lipoproteins (VLDL) in the cockerel was studied both in vivo and in vitro. Synthesis was studied by immunoprecipitation techniques with antisera prepared against VLDL and a major VLDL protein. VLDL were isolated from the plasma of white Leghorn hens and estrogen-treated white Leghorn cockerels by ultracentrifugal flotation at d 1.006 g/ml. After delipidation, the lipid-free proteins (apoproteins) were fractionated on Sephadex G-150 and DEAE-cellulose. Both the hen and the estrogen-treated cockerel VLDL were shown to contain an identical apoprotein with a mol wt of approximately 12,000; the apoprotein is designated fraction B. Reduction and S-carboxy-methylation of fraction B resulted in a reduction of the molecular weight by approximately one-half, indicating a dimer-monomer relationship. Antiserum prepared to the hen VLDL dimer protein gave precipitin lines of complete identity to both the hen and cockerel dimer, monomer, VLDL, apoVLDL, low density lipoproteins, and plasma; no precipitin line was formed with either hen or cockerel high density lipoproteins. After a single subcutaneous injection of diethylstilbestrol into the cockerel, plasma VLDL protein, cholesterol, and triglyceride increased, reaching a maximum 24--48 h after hormone administration. Liver slices from similarly treated animals were incubated in vitro in culture medium in the presence of [3H]lysine for 2 h. Immunoprecipitable radioactivity in VLDL increased within 2 h of diethylstilbestrol treatment and reached a maximum at 24 h; VLDL radioactivity returned to base-line levels by 72 h. At the peak of induction, newly synthesized VLDL represented 11% of the total soluble protein synthesized. When actinomycin-D (5 mg/kg) was administered simultaneously with estrogen, the induction of VLDL synthesis was totally inhibited. To determine whether the effect of estrogen on VLDL synthesis was mediated at the level of transcription, partially-purified cockerel liver mRNA was prepared from estrogen-treated animals and the mRNA activity for fraction B was quantitated in a wheat germ translation system. Fraction B mRNA was found to increase from a low base-line value to a maximum 16-24 h after estrogen treatment, returning towards baseline values at 30 h. At the peak of induction, fraction B constituted 12% of the total protein synthesized. The kinetics of induction of fraction B mRNA activity in the cell-free translation system is very similar to that observed in liver slice experiments. This finding suggests that estrogen stimulates VLDL synthesis, at least partially, by enhancing the accumulation of the mRNA for one of their major apoproteins.

PIP: In vivo and in vitro studies were undertaken to investigate the mechanism of the induction of the synthesis of very low density lipoproteins (VLDL) by estrogens in the cockerel. VLCL were isolated from plasma of white Leghorn hens and estrogen-treated white Leghorn cockerels. VLCL from both these groups contained an identical apoprotein (Fraction B) with a molecular weight of about 12,000. Reduction and S-carboxy-methylation of this fraction reduced its molecular weight by approximately 50%, thus indicating a dimer-monomer relationship. When antiserum was prepared against the hen VLDL dimer protein, completely identical precipitin lines were found for both the hen and cockerel dimer, monomer, VLDL, apoVLDL, low density proteins, and plasma. However, no precipitin line was formed with hen and cockerel high density lipoptoteins. A single sc injection of diethylstilbestrol (DES) into the cockerel increased levels of plasma VLDL protein, cholesterol, and triglyceride, with maximum values occurring 24-48 hours after injection. Immunoprecipitation of liver slices from similarly treated animals showed an increase of radioactivity of VLDL within 2 hours of injection. Values reached a maximum at 24 hours and returned to baseline levles by 72 hours. Newly synthesized VLDL comprised 11% of the total soluble protein synthesized during the period of peak values. Actinomycin-D (5 mg/kg), when administered simultaneously with the estrogen, completely inhibited the induction of VLDL synthesis. In another experiment, partially purified cockerel liver mRNA was prepared from estrogen-treated animals and the mRNA acitivity for Fraction B was measured in a wheat germ translation system. Values for Fraction B mRNA reached a maximum 16-24 hours after estrogen-treatment and returned to baseline levels by 30 hours. Fraction B represented 12% of the total protein synthesized at the peak of induction. The results suggest that estrogen stimulates the synthesis of VLDL by enhancing the accumulationg of the mRNA of 1 of their major components.

Long-term vasectomy: effects on the occurrence and extent of atherosclerosis in rhesus monkeys.

We demonstrated previously that atherosclerosis develops more extensively in vasectomized cynomolgus macaques fed an atherogenic diet and speculated that the immunologic response to sperm antigens may have exacerbated the atherosclerosis. We report here that rhesus monkeys vasectomized for 9-14 yr and fed monkey chow (devoid of cholesterol and low in fat) rather than an atherogenic diet also had more extensive and severe atherosclerosis than did control animals of the same age. The extent of atherosclerosis was considered as the percentage of intimal surface with plaques. No control animals were found to have plaques in the thoracic aorta, but 7 of 10 vasectomized monkeys were affected. The plaques in the vasectomized monkeys occupied about 13% of the intimal surface. In 4 of 7 control monkeys and 7 of 10 vasectomized monkeys there were lesions in the abdominal aortas; the lesions were considerably more extensive and severe in the vasectomized animals. Lesions were also more common in iliac arteries of vasectomized animals, and the extent was increased about threefold. Plaques were seen at the carotid bifurcation in all of the animals of both the control and vasectomized groups. The carotid bifurcation plaques of the vasectomized monkeys were larger than those of the control animals on the right but not on the left side. Histologically, the lesions of vasectomized monkeys did not appear to be qualitatively different from those of control animals, even though they were larger and contained more collagen, lipid, and mucopolysaccharides. Grossly, the distribution of the lesions in the vasectomized animals was different from that in the control animals, and that of lesions induced by atherogenic diets, i.e., the lesions were distributed randomly within the artery rather than around bifurcations. More extensive atherosclerosis was noted among vasectomized animals that were found to lack demonstrable circulating free antisperm antibodies. On the basis of the observations made in this study, we suggest that the antisperm antibodies that form after vasectomy may result in circulating immune complexes that exacerbate atherosclerosis.

[Effects of T helper 1 cells and T helper 17 cells secreting cytokines on rat models of experimental periodontitis].

Objectvie: To investigate the effects of secreting cytokines interferon-gamma (IFN-γ) and interleukin-17 (IL-17) of T helper 1 cells (Th1) and T helper 17 cells (Th17) on the peripheral blood and alveolar bone destruction, so as to provide a new explanation for cellular immunity-mediated alveolar bone destruction. Methods: Eighteen eight-week-old male Sprague-Dawley rats were divided, randomly and equally, into 3 groups: lipopolysaccharide (LPS) group, ligation group and normal control group. In the LPS group, Escherichia coli LPS was injected into the alveolar mucosa on the buccalmedian site of the left upper first molar, while the right upper first molar was injected with equal volume of physiological saline as self-controls. The injections were performed every other day for four times totally. In the ligation group, the left upper first molars were ligatured with 0.2 mm orthodontic cords, while the right upper first molars were left untreated as self-controls, and supplemented with high-sugar diet to promote the periodontitis status. The rats in normal control group were fed normally. The concentrations of IFN-γ and IL-17 in peripheral blood were measured using enzyme linked immunosorbent assay (ELISA) method at the fourth week after the start of injection and at the eighth week after ligation. The histological of periodontal tissues were observed after hematoxylin-eosin (HE) staining and osteoclast count was performed under light microscope. The histological of osteoclasts were observed after tartrate-resistant acid phosphatase (TRAP) staining. Expression of IFN-γ and IL-17 were detected by immunohistochemical assay. Results: The concentrations of IFN-γ in peripheral blood of LPS group [(185.0±50.7) ng/L] and ligation group [(202.9±60.4) ng/L] were significantly higher than that of normal control group [(106.3±17.2) ng/L](P<0.05). Meanwhile, histological examination showed inflammatory cells infiltration in the gingival epithelium, the height reduction of alveolar bone accompanied with absorption lacuna. There were significantly higher HE and TRAP stained osteoclasts in LPS group (9.50±1.05) and ligation group (10.83±1.17) than that in controlgroup (0.33±0.52)(P<0.05). Moreover, the expressions of IL-17 in alveolar bone absorption area of LPS group and ligation group were significantly stronger than that in control group (P<0.05). Conclusions: The rat models of experimental periodontitis and alveolar bone resorption could be successfully established by means of ligationand LPS injection, respectively. The periodontal inflammatory responses were related to secreting cytokines IFN-γ and IL-17 of Th1 and Th17 cells, while Th17 cells might exert a positive effect on alveolar bone destruction.

Efecto contráctil del extracto acuoso de Artemisia absinthium (ajenjo) frente a oxitocina en útero aislado de ratas / Contractile effect of aqueous extract of Artemisia absinthium (ajenjo) against oxytocin in isolated uterus of rats

RESUMEN Introducción. El uso de Artemisia absinthium (ajenjo) en el trabajo de parto es ampliamente empleado en países de la región; sin embargo, no hay evidencia científica suficiente de su eficacia y seguridad, lo que representa un alto riesgo maternofetal. Objetivo. El propósito fue evaluar el efecto contractil de la Artemisia absinthium (ajenjo) en comparación a la oxitocina en útero aislado de ratas. Métodos. Se separaron 8 ratas Holtzman en i) Grupo experimental: Artemisia dosis 5, 10, 20, 30, 40 y 50 mg y ii) Grupo control: oxitocina 10-6 M. Se montaron los úteros en un baño de órganos aislados y se registraron contracciones por 5 minutos. Resultados. La frecuencia de contracciones con ajenjo 40 y 50 mg fueron comparables con oxitocina (p>0,05). Asimismo, dosis de 20 y 30 mg de Artemisia provocaron contracciones significativamente más duraderas que la oxitocina. La intensidad resultó ser comparable con oxitocina con dosis de 20, 40 y 50 mg de Artemisia. Conclusión. El extracto acuoso de Artemisia absinthium presentó efecto contráctil similar a la oxitocina y dependiente de la dosis en útero aislado de ratas.

ABSTRACT Introduction. The use of Artemisia absinthium (wormwood) in labour is widely used in countries in the region; however, there is no enough scientific evidence of its efficacy and safety, which represents a high maternalfetal risk. Objective. The purpose was to assess the contractile effect of Artemisia absinthium (wormwood) compared to oxytocin in utero isolated from rats. Methods. Eight Holtzman rats were separated into i) Experimental group: Artemisia dose 5, 10, 20, 30, 40, and 50 mg and ii) Control group: oxytocin 10-6 M. The uteruses were mounted in an isolated organ bath, and contractions were recorded for 5 minutes. Results. The frequency of contractions with wormwood 40 and 50 mg were comparable with oxytocin (p>0.05). Likewise, doses of 20 and 30 mg of Artemisia caused significantly longer-lasting contractions than oxytocin. The intensity was found to be comparable with oxytocin at doses of 20, 40, and 50 mg of Artemisia. Conclusion. The aqueous extract of Artemisia absinthium has a contractile effect similar to oxytocin and is dose dependent in utero isolated from rats.

General criteria for assessing the evidence for carcinogenicity of chemical substances: report of the Subcommittee on Environmental Carcinogenesis, National Cancer Advisory Board.

PIP: The evaluation of the potential human hazards of a given agent must be individualized in terms of the chemical and metabolic aspects of the agent, its intended use, and the data available at the time. An agent is carcinogenic in man if it increases the incidence of malignant neoplasms, or a combination of benign and malignant neoplasms in humans to levels that are significantly higher than those in a comparable group not exposed to the agent. Types of evidence suggesting that an agent is carcinogenic include neoplastic response directly related to exposure, and incidence and mortality differences related to occupation exposure or geographic regions. Negative epidemiologic data may not establish the safety of suspected materials. The carcinogenicity of a substance is also established when the administration to groups of animals in adequate experiments results in increases in the incidence of 1 or more types of malignant neoplasms in the treated compared to the control groups. The demonstration that the occurrence of neoplasms is dose-dependent also qualifies as a positive result. If a substance is found to induce benign neoplasms it should be considered a potential health hazard. Some short-term or in vitro tests which appear promising include assays for the induction of DNA damage and repair, mutagenesis in bacteria, yeast, and neoplastic transformation of mammalian cells in culture. Quantitative extrapolation from animal studies to evaluate human risks is not a clear science; each case must be considered individually. When a compound has been proven carcinogenic there remains the question of the extent to which the possible risks to man are counterbalanced by the possible benefits of the substance.^ieng

Tissue sites of alpha fetoprotein synthesis by the rat during pregnancy and hepatoma growth.

The tissue sites of alpha1 fetoprotein (AFT) synthesis by the rat during gestation and hepatoma growth were determined by specific incorporation of a radiolabeled amino acid precursor into AFP by tissue cultures in vitro. During gestation, AFP were produced by the yolk sac, the fetal liver, and in small amounts by the fetal gastrointestinal tract; there was no synthesis by maternal rat tissues. During growth of a transplantable hepatoma, only the hepatoma tissue synthesized AFP; the nontumor tissue of the host contained AFP but did not produce it.

PIP: Alpha fetoprotein (AFP) synthesis by adult rats during gestation and hepatoma growth was determined in vitro with specific precipitations of radiolabeled AFP antisera after incubation of Spinner cultures of various rat tissues in arginine-free culture medium containing radiolabeled arginine. In general, AFP was synthesized by fetal liver, yolk sac, small intestine, and transplantable (tumor) tissue; none of the normal adult tissues, including testis or ovary, produced AFP. AFP synthesis (measured over 22 hours) was confined to the fetal liver (367 ng), yolk sac (1,368 ng), and to a small extent, the gastrointestinal tract during 19-day gestation. None of the maternal tissues produced AFP. When measured during growth of a transplantable hepatoma, AFP was synthesized only by the hepatoma tissue, though the nontumor tissue of the host contained AFP, due to release of AFP from the cultured tissue as it degenerated in vitro, but did not produce it (noninvolved tissues of hepatoma-bearing rats did not incorporate labeled arginine into AFP in vitro). Identifying fetal organs responsible for AFP synthesis explains observed AFP concentration changes in the postpartum period in rats, since elevated AFP in the mother is caused by AFP produced by the fetus which crosses the placenta or yolk sac to maternal circulation. Elevations above normal (.06 mcg/ml) adult rat concentrations occur in 3 circumstances in the nonpregnant rat: 1) development of AFP-producing tumors; 2) proliferation by normal liver cells; and 3) exposure to chemical carcinogens.

Hormone-dependent mammary tumors in strain GR/A mice. II. Preneoplastic and neoplastic properties.

In a series of transplant experiments, we investigated interactions between normal, ductal phase, and tumorous phase mammary tissues in GR/A mice, with particular respect to growth regulation. Transplants from hormone-dependent mammary tumors (HDT) transplanted into mammary fat pads already containing normal mammary ducts usually could not be located subsequently or, at best, displayed minimal growth. Growth regulation was also normal when two HDT transplants, placed in a single gland-free fat pad in nonpregnant hosts, produced ductal outgrowths displaying mutual avoidance behavior in which ducts did not touch and were normally spaced. A normal and HDT transplant in a single gland-free fat pad also showed identical, normal regulatory behavior. In contrast, HDT transplants in pregnant hosts or in hosts receiving exogenous hormone therapy displayed altered growth patterns in which neighboring tumors touched and eventually fused. When surrounded by normal tissues, the HDT continued to proliferate and often appeared to overgrow and engulf normal elements. We concluded that HDT tissues grown in nonpregnant hosts fully responded to those short-range regulatory influences characteristic of normal morphogenesis. When exposed to hormones of pregnancy, however, these interactions changed, and HDT tissues exhibited many characteristics of ductal carcinomas.

PIP: A series of transplant experiments, designed to investigate interactions between normal, ductal phase mammary tissues in GR.A mice, with particular emphasis on growth regulation, is reported. Hormone-dependent mammary tumors (HDT) were transplanted into the number 4 fat pad of either virgin or pregnant hosts. Normal mammary tissue was also transplanted. Transplants for HDT into fat pads already containing normal mammary ducts displayed minimal growth. Growth regulation was also normal when 2 HDT transplants, placed in a single gland-free fat pad in nonpregnant hosts produced ductal outgrowths displaying mutual avoidance behavior. Normal regulatory behavior was also exhibited when abnormal and HDT transplant were placed in a single gland-free fat pad. However, HDT transplants placed in pregnant hosts or in hosts receiveing exogenous hormone therapy, altered growth patterns were seen with the tumors touching and eventually fusing. When surrounded by normal tissues, the HDT continued to proliferate, overgrow and engulf normal tissue. When exposed to the hormones of pregnancy, the normal regulatory influences are superceded, and HDT tissues exhibit characteristics of ductal carcinoma.

Direct action of estradiol on rat mammary tumors.

PIP: This study reports a direct action of 17-beta-estradiol on protein synthesis by 7, 12-dimethylbenz (alpha) anthracene (DMBA) induced rat mammary tumors. Sprague-Dawley female rats were given 20 mg of DMBA in sesame oil by gastric tube. Mammary tumors developed. When tumors reached 1.5 to 2 cm in diameter, animals were ovariectomized. At 4-7 days later animals were killed and tumors removed. Microscopic examination confirmed the tumors to be carcinoma of adenoid cystic variety with regressive changes. 14 tumors from 14 animals were found suitable for study. In vitro treatment with 17-beta-estradiol gave rise to a 7% increase in the rate of 3H-leucine incorporation, expressed as dpm/mg of TCA-insoluble protein. This increase was considered statistically significant (p.001). A large variation among different tumors was noted. Also results varied with differences in time after ovariectomy. Under the same incubation conditions the same percentage of increase in the rate of 3H-leucine incorporation had been observed in the study of the effect of estrogen on the uterus of ovariectomized rats. In other mammalian tissues studied those containing high levels of estrogen receptor were able to respond to direct stimulation of estrogen. It was concluded that estrogen directly stimulates protein synthesis in the mammary tumors. This supports the view that these tumors are estrogen responsive tissues.^ieng

Autoradiographic localization of prolactin receptors in 7,12-dimethylbnez[a]anthracene-induced rat mammary carcinoma.

Prolactin receptors were localized by autoradiography in 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumors from rats by incubation of tumor slices with [125I]ovine prolactin. Specific prolactin binding was confined to tumor cells, and nonspecific binding was present in alveolar spaces and connective tissue. In some tumors, all cells contained receptors; in others, up to one-half the cells remained unlabeled. These results suggested that variation in receptor content in DMBA-induced mammary tumors may be caused by two distinct populations of cells--one which contains receptors and another which possesses very few receptor sites or none at all.

PIP: In this investigation, prolactin receptor sites were localized by autoradiography in 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumors from rats. Tumors were removed when they reached the size of 2 X 4 cm and sliced to a thickness of .5 mm. Sections cut from the slices were incubated for 3 hours in medium 199 (1% bovine serum albumin, 5 mM CaC12, 5mM HEPES buffer) at pH 7.4 and 400,000 counts/minute of iodine-125-ovine prolactin in the presence or absence of 1 mcg of unlabeled prolactin. After further preparation and storage for 4 months in a light-tight box slides were developed, fixed and stained and then photographed. All tumors were labeled by iodine-125-ovine prolactin. Prolactin receptors were associated with only half the total cells. Specific prolactin binding was confined to tumor cells and nonspecific binding was present in alveolar spaces and connective tissue. In some tumors prolactin recepotrs were found in all tummor cells, whereas in other tumors half of the cells did not contain any receptors, or very few. Reproductions of autoradiographs illustrate findings. Results suggest that variations in receptor content of the tumors may be caused by 2 distinct populations of cells. 1 type contains many receptors and the other very fewreceptor sites, or none at all.

Enhancement of tumor growth and metastases by medroxyprogesterone acetate in transplanted uterine adenocarcinoma cells of the rat.

PIP: This study describes the effects of medroxyprogesterone acetate on tumor growth and metastases in transplanted uterine adenocarcinoma cells of the rat. High-and-low-tumorigenic cloned cells of Sprague-Dawley rat uterine adenocarcinoma originally induced by 7,12-dimethylbenz (alpha) anthracene in vivo were used. Both were derived from the same parent culture. They were cultured for more than 2 years and both retained almost the same transplantability. Survival rate of cell colonies in vitro was reduced in both lines after progesterone treatment of more than 8 mcg per ml. This reduction was dose dependent. About 1 million cells suspended in .2 ml culture medium were injected sc into the interscapular region of isologous newborn rats. At 5 weeks these rats were given .5 mg medroxyprogesterone acetate twice a week for 2 weeks. At 7 weeks they were killed. High-tumorigenic cells produced growing tumors in all newborn rats. About a third of these rats died of metastases during the 7-week observation period. Tumors produced by low-tumorigenic cells grew slowly and occasionally regressed without metastases to the lung. Tumors in female rats were larger than those in males. Enhancement of tumor growth and metastases by this progesterone compound was observed in rats inoculated with low-tumorigenic cells as compared to controls. The enhancement was not significant in tumors produced by high-tumorigenic cells. The progesterone may act immunosuppressively in vivo, or make alterations in environmental conditions of the tumors.^ieng

Mammary nodules in dogs during four years' treatment with megestrol acetate or chlormadinone acetate.

PIP: A 7 year study of megestrol and chlormadinone in female dogs is in progress. This report characterized histopathologically 60 mammary nodules during the first 4 years of the study. 100 purebred female beagles, 6-12 months of age, were randomly assigned to 5 equal groups. One group was used as a control. Oral doses were .01, .10, and .25 mg/kg/day of megestrol acetate in coconut oil in capsules and of chlormadinone acetate .25 mg/kg/day in lactose tablets. These doses were 1, 10, and 25 times the projected dose of megestrol for humans and about 25 times the human dose of chlormadinone. After 2 years 4 dogs from each group were necropsied. One high-dose megestrol-treated and 1 chlormadinone-treated dog had benign mixed mammary tumors. Palpable nodules were first observed at 16 months in the chlormadinone-treated dogs, at 18 months in dogs given the high dose megestrol and at 27 months in the dogs treated with middle-dose megestrol. Transitory nodules were found in 4 control dogs after 21 months and in low dose megestrol-treated dogs at 26 months. Of 38 grossly detected nodules evaluated microscopically from the megestrol-treated dogs 27 were nodular hyperplasia, 5 were benign mixed mammary tumors, 3 were ductal dialatations, 1 was a lymph node, 1 was fat necrosis and 1 was the umbilicus. Of 22 nodules from the chlormadinone-treated dogs 12 were nodular hyperplasia, 4 benign mixed mammary tumors, 1 chondromucoid degeneration and 1 adenocarcinoma with widespread metastases. 3 nodules were lymph nodes and 1 other had no mammary tissue. Involutions, regression and sclerosis of many areas of nodular hyperplasia were evident at 4 years. Thus of the 60 nodules evaluated during the first 4 years of the study 50 were non-neoplastic and 10 were neoplastic. It is considered that the 1 adenocarcinoma may have been spontaneous and not a treatment-related neoplasm. A precursor stage through nodular hyperplasia apparently did not occur.^ieng