Experimental Biology and Medicine

Aflatoxin B₁ (AFB₁) is a fungal toxin that has been implicated as a causative agent in human hepatic and extrahepatic carcinogenesis. In this review, the mechanisms involved in AFB₁ toxicity are delineated, in order to describe the features that make a specific cell, tissue, or species susceptible to the mycotoxin. Important considerations include: (i) different mechanisms for bioactivation of AFB₁ to its ultimate carcinogenic epoxide metabolite; (ii) the balance between bioactivation to and detoxification of the epoxide; (iii) the interaction of AFB₁ epoxide with DNA and the mutational events leading to neoplastic transformation; (iv) the role of cytotoxicity in AFB₁ carcinogenesis; (v) the significance of nonepoxide metabolites in toxicity; and (vi) the contribution of mycotoxin-unrelated disease processes. Although considerable controversy remains about the importance of specific events, a great deal has been learned about biochemical and molecular actions of AFB₁.

Top-down elasticity analysis is a novel extension of top-down metabolic control analysis. It has provided researchers with a theoretical and practical platform upon which quantitative analyses of the sites of action of hormones and drugs can be based. This approach is easy to apply and involves dividing up the metabolic system in question into two or three blocks of enzyme reactions around an intermediate between the blocks of reactions. The kinetic response of each block to the intermediate is measured in the steady state in situ by determining the flux through the block at different measured intermediate concentrations. The intermediate can be manipulated by titrating the other blocks with suitable inhibitors or activators. Then, to determine which blocks of reactions are quantitatively the most important in terms of any change in the flux rate of the system, a quantitative comparison of the titration curves from the experimental preparations is made with those of the control preparations. In this minireview we will examine, as an example, the use of top-down elasticity analysis for the quantitative identification of the important sites of action of thyroid hormones on oxidative phosphorylation in hepatocytes. The experimental results show that approximately 50% of the change in resting oxygen consumption in hepatocytes from hypothyroid and hyperthyroid rats (compared with euthyroid controls) is attributable to changes in the rate of the mitochondrial proton leak; the remaining 50% is accounted for by changes in nonmitochondrial- and ATP turnover-dependent oxygen consumption in hypothyroid and hyperthyroid hepatocytes, respectively.

Recent developments in magnetic resonance (MR) imaging have opened up new avenues in the investigation of cardiovascular physiology. Inherent signal intensity of any tissue on MR images depends largely on proton concentration as well as longitudinal (T1) and transverse (T2) relaxation times. Myocardial contrast can be manipulated by using specific MR pulse sequences which are selectively sensitive to differences in any one of these parameters. Paramagnetic metal complexes are used as contrast media in MR imaging to enhance the inherent contrast. Contrast media in MR imaging are not directly visible but change the magnetic properties of other nuclel in close proximity, such as those of the water hydrogen. The signal of water can be altered by the contrast medium in different ways, either by changing the relaxation times or through bulb susceptibility effects, or both.

The role of MR contrast media for quantitative characterization of ischemic heart disease has advanced considerably in the past 10 years. Conventional MR imaging techniques following the administration of contrast media are useful for identifying and sizing myocardial infarctions and for distinguishing between occlusive and reperfused myocardial infarctions as well as reversible (stunned) and irreversible injuries. Recent results suggest that contrast-enhanced MR imaging can also be used to identify dead cells in reperfused ischemically injured myocardium.

The recently developed fast MR imaging techniques, with the aid of MR contrast media as a perfusion indicator, may be useful in estimating regional myocardial perfusion and blood volume. The assessment of capillary circulation or myocardial perfusion may be used for evaluating the extent of hypoperfusion and treatment efficacy. Experimental and clinical perfusion studies indicate that perfusion-sensitive MR imaging detects compromised myocardium (area at risk). Combining myocardial perfusion imaging with the anatomic and functional information provided by other MR imaging sequences could make MR imaging a comprehensive noninvasive technique for the evaluation of ischemic heart disease.

Chloride (Cl^-) channels were characterized in vascular smooth muscle cells (VSMC) using radioisotope flux and patch-clamp electrophysiological techniques. Transmembrane ¹²⁵iodine (¹²⁵l) efflux from subcultured (Passage 1-5) rat aortic VSMCs was used as an indicator of Cl^- movements to study the relationship between intracellular calcium concentration ([Ca²⁺]₁) and CI^- channel activity. Angiotensin II (Ang II) (10^-7 M) and adenosine 5'-triphosphate (ATP) (10^-4 M) induced rapid increases (9,7- and 14.9-fold, respectively) in ¹²⁵I efflux rates. We found that both Ang II-and ATP-stimulated ¹²⁵I efflux and [Ca²⁺]_I increases were completely abolished after brief incubation (20 μM, 20 min) with the acetoxymethyl ester of 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM), a membrane-permeable Ca²⁺ chelator. However, when external EGTA was used to blunt agonist-stimulated Ca²⁺ influx, ¹²⁵l efflux was still increased in response to Ang II and ATP. These data suggest that Ca²⁺ release from intracellular sites is sufficient to activate CI^- channels in response to Ang II and ATP. Using standard patch-clamp electrophysiological techniques, we found that Ang II, a Ca²⁺-mobilizing agonist, stimulated outward Cl^- currents (g_cl = 75 pS) in cell-attached (C/A) patches of primary and subcultured VSMCs. Collectively, these data suggest that Ang II and other vasoconstrictor agents stimulate Cl^- channel activity via increases in [Ca²⁺]_l. Cl^- channel activation may help to depolarize the VSMC membrane leading to increased Ca²⁺ influx during agonist stimulation.

Physicochemical studies have suggested that the 43-kDa plasma membrane fatty acid binding protein (FABP_pm) is closely related to the mitochondrial isoform of aspartate aminotransferase (mAspAT). In the present studies, mAspAT was not detected immunohistochemically or by immunoblotting in plasma membranes of proliferating 3T3-L1 fibroblasts. During controlled differentiation to an adipocyte pheno-type, mAspAT became detectable by the second day of confluent growth, prior to accumulation of visible lipid droplets, and was strongly expressed in 8-day differentiated 3T3-L1 adipocytes. The pattern of expression paralleled the previously reported expression both of FABP_pm and of the V_max for saturable uptake of long chain free fatty acids. As with anti-FABP_pm, antibodies to mAspAT selectively inhibited the uptake of [³H]-oleate in 3T3-L1 adipocytes but not in fibroblasts, while having no effect on uptake of either 2-deoxyglucose or the medium chain fatty acid octanoate. Preabsorption of anti-FABP_pm with mAspAT, or of anti-mAspAT with FABP_pm, abolished immunopositivity in immunohistochemical and immunoblotting studies, as well as the ability of either antibody to inhibit [³H]-oleate uptake. These studies provide strong biologic evidence for the identity of FABP_pm and mAspAT, and for the hypothesis that FABP_pm/mAspAT mediates the uptake of long chain free fatty acids.

There is considerable evidence suggesting a role for the macrophage (Mφ) in the development of glomerulosclerosis (GS) and atherosclerosis, lesions which appear to be analogous. Migration of mesangial cells (MC), which are modified smooth muscle cells, may play a role in the pathogenesis of glomerular injury, and smooth muscle migration may play a role in the pathogenesis of atherosclerosis as well. We undertook the present study to determine the effects of Mφ supernatants (MφSN) on MC migration and formation of MC hillocks, which are considered an in vitro model of GS. By means of a migration assay using wounded cultures of confluent, growth-arrested MC, MC migration was found to be significantly enhanced by incubation with MφSN at 24 hr (migration score: MφSN, 24.3 ± 1.3; control, 11.6 ± 1.0, P < 0.001) as well as 48 hr incubation (migration score: MφSN, 34.0 ± 1.4; control, 15.4 ± 1.4, P < 0.001). Enhanced MC migration following prolonged incubation with MφSN was also shown using phase contrast microscopy and scanning electron microscopy. MC hillock formation was enhanced by MφSN in a concentration-related manner as was hillock size. These data demonstrate that MφSN can directly enhance MC migration and hillock formation, processes that may in part account for the observed role for the Mφ in the development of mesangial expansion and GS as well as atherosclerosis.

Genetic variation in fetal survival, maternal plasma androgen levels, and ovarian aromatase activity was examined mid (Day 9) and late gestation (Day 16) in strains of mice that differ in reproductive performance (A/J, C57BL/6J, C8/Jls, C17/Jls, and S15/Jls). At both gestational stages, females selected for large litter size (S15/Jls) carried more fetuses than any of the other strains examined. Particularly at midpregnancy, S15/Jls females also maintained higher plasma levels of androstenedione and testosterone relative to both control strains, C8/Jls and C17/Jls. Consistent with previously reported changes in peripheral estrogen levels during mouse pregnancy, aromatase activity was higher on Day 16 than on Day 9. This study demonstrates genetic variation in fetal survival that is correlated with increased maternal androgen levels. A stage-specific gestational increase in aromatase activity occurs in several strains of mice and is associated with elevated plasma estrogen during the second half of pregnancy.

Previous studies have shown that target tissues cleave rat prolactin (rPRL) to form a two-chain derivative that yields ∼ 16- and ∼ 6-kDa fragments upon reduction. Both cleaved rPRL and the purified 16-kDa fragment have novel biological activities. Thus, cleavage may be of physiological significance. We determined whether normal mammary tissue or stroma (lacking mammary epithelial cells [MEC]) or mammary tumor tissue could cleave rPRL in vitro. Tumor explants were derived from a transplantable mammary tumor cell line (TPDTMT-4EP). The hormone was incubated with explants of those tissues from female BALB/c or DDD mice. Medium samples were processed by SDS-PAGE, and the relative abundance of intact and cleaved rPRL was determined by densitometric analysis of immunoblots using an antiserum to the 16-kDa fragment of rPRL.

After 2-hr of incubation, normal DDD mammary explants cleaved ∼ 25% of added rPRL, but tumor explants cleaved none. Explants of intact virgin BALB/c mouse mammary gland and of parenchyma-free “cleared” mammary fat pad cleaved rPRL equally well, but explants of abdominal adipose tissue had low cleaving activity. Homogenates of cleared mammary fat pads that were incubated with rPRL contained a high proportion of the cleaved form but none of the free 16-kDa fragment. These results indicate that cleavage of rPRL is highly specific to mammary stroma. Such cleavage may yield a form of the hormone that has novel activities within the mammary gland or elsewhere in the body.

The effects of treatment with a perfluorochemical emulsion plus carbogen on radiation pneumonitis were examined in a rat model system. Rats received thoracic irradiation (15 Gy) and radiation reactions in the lungs were assessed 25 and 35 days later using bronchoalveolar lavage and histologic assessments. The irradiated lungs showed the expected evidence of acute radiation pneumonitis, including protein leaks and also alveolar infiltrates and interstitial infiltrates. Administration of a perfluorochemical emulsion (Fluosol; 15 ml/kg) plus carbogen breathing for 30 min before and during irradiation did not enhance the reactions seen in the irradiated lungs.

We recently reported that the antitumor activity of an immobilized oxidase-peroxidase system, which can produce radical species as intermediates, is dependent on the presence of an intact and functioning immune system in the host. A number of recent investigations have indicated that the bioactive form of many quinone-type anticancer agents may be the semiquinone-type radical. To investigate if the antitumor activity of some of the quinone-type anticancer agents may also be dependent on a fully functioning immune system in the host, we used as a simple, well-defined model the 2,6-dimethoxybenzo-p-semiquinone radical. This free radical has a potent antitumor activity against Ehrlich ascites tumors in mice, as first described by Szent-Gyorgyi's group. In the present study, we investigated its antitumor activity in immunocompromised BALB/c mice.

We found that the antitumor activity of the semiquinone radical is completely abolished in mice that had previously been subjected to whole body γ irradiation, indicating a possible involvement of one or more cells of the host's defense system. The antitumor activity was not abolished in mice with dysfunctional macrophages induced by a severe selenium deficiency. However, the radical was inactive in mice whose immune system was suppressed with cyclosporin A and in nude (nu/nu) mice.

These results suggest that the dimethoxybenzo-semiquinone radical, rather than acting primarily as a cytotoxic agent as previously reported, exerts its antitumor activity mainly via a more complex mechanism in which T lymphocytes appear to play a major role.

Hyperinsulinemia may link diabetes to hypertension. We evaluated the effects of insulin on blood glucose and blood pressure in control and streptozotocin (STZ)-induced (60 mg/kg, iv) diabetes. Insulin was given daily for 10-14 weeks to both diabetic (0, 1, 2, and 3 U) and control (0, 2, and 3 U) male rats (n = 7-8/group). Indirect and direct blood pressures were measured as were blood glucose and several metabolic parameters. All treated rats became hyperinsulinemic. Untreated and 1 U insulin-treated diabetic rats were hyperglycemic. Higher doses of insulin significantly reduced blood glucoses in diabetic animals. Indirect blood pressure measurements were unchanged between groups. However, when measured directly, the untreated diabetic rats had significantly lower pressures that did untreated controls. Insulin treatment at dosages above 1 U normalized blood pressure in diabetic animals. These same doses of insulin also restored to normal all the metabolic parameters associated with diabetes. Insulin treatment did not affect any of the parameters evaluated in nondiabetic rats. Collectively, the results show that STZ-induced diabetes results in a decrease in blood pressure and that insulin treatment can restore blood pressure in diabetic rats. Although the results suggest that insulin may be involved in restoring blood pressure in animals with a carbohydrate imbalance, the precise mechanism for elevating blood pressure is not known with certainty.

Phytoestrogens found in clover, alfalfa, and soybeans have caused reproductive toxicity in several mammalian species. Other estrogens, such as diethylstilbestrol (DES), are developmental toxicants, reducing uterine estrogen receptor (ER) concentration, altering uterine growth, and eliciting reproductive tract abnormalities in the rat. The present study examines the effects of the phytoestrogens coumestrol and equol on the developing rat uterus. Various doses of these compounds were injected sc on postnatal days (PND) 1-5 or 1-10 to ascertain their effects on uterine weight and ER levels, and on PND 10-14 to determine their effects on uterine weight and gland genesis. Coumestrol (PND 1-5) was about 10^-3 as potent as DES in increasing uterine weight (wet or dry) while equol increased dry weight only, with a potency of 10^-5 that of DES. Although the 10 and 100 μg doses of coumestrol (PND 1-5 or 1-10) initially increased uterine wet weight, by PND 20 uterine weights either equaled or fell significantly below controls. The 100-μg dose of coumestrol (PND 1-5 or 1-10) reduced ER levels at all ages, while the 10-μg dose was not as effective. Equol (PND 1-5 or 1-10) did not affect ER levels. Premature uterine gland genesis occurred by PND 9 for the PND 1-5 100-μg coumestrol dose. When given on PND 10-14 (the critical period of gland genesis), 10 μg and 100 μg of coumestrol and 10 μg DES greatly increased uterine weight, while no effect was elicited by equol. Although coumestrol and equol inhibited uterine gland genesis in a dose-dependent manner, neither abolished gland genesis as did 10 μg of DES or tamoxifen. These data demonstrate that coumestrol elicits uterine biochemical and morphological toxicity much like DES. Equol decreased uterine gland number without increasing uterine wet weight or luminal epithelial hypertrophy, which is inconsistent with either an estrogenic or antiestrogenic action in the uterus.