John R. Trevithick

Vitamin E Intake and Risk of Cataracts in Humans

Experimental evidence suggests that oxidative stress due to the accumulation of free radicals plays a role in the pathogenesis of cataracts and that the process can be prevented or ameliorated by antioxidants. In addition, a recent study found that cataract patients tended to have lower serum levels of vitamins C, E, or carotenoids than did control subjects. This investigation, which compared the self-reported consumption of supplementary vitamins by 175 cataract patients with that of 175 individually matched, cataract-free subjects, revealed that the latter group used significantly more supplementary vitamins C and E (p = 0.01 and 0.004, respectively). Inasmuch as the observed reduction in risk of cataracts was at least 50%, a randomized, controlled trial of vitamin supplementation in cataract prevention seems justified.

Topical tocopherol acetate reduces post-UVB, sunburn-associated erythema, edema, and skin sensitivity in hairless mice

Exposure of the skin of the back of skh-1 hairless mice to UVB (310 nm peak) irradiation at doses of 0.115-0.23 J/cm2 results after 24-48 h in an erythema which can be quantified using an erythema meter, providing a useful model of sunburn. Application of pure d-alpha-tocopherol acetate, a thick oil, to the skin immediately following the exposure to UVB significantly reduces the increase in erythema index, by 40-55%. At the lower dose (0.115 J/cm2), skin thickness (associated with edematous swelling of the sunburned skin) was measured by a novel non-invasive technique not previously reported for this purpose--magnetic resonance imaging (MRI). In two experiments the UVB-induced increase in skin thickness was significantly reduced at 24 hr by 29 and 54%, and at 48 hr by 26 and 61%. After 8 days the untreated irradiated mouse skin still showed a significant increase in thickness (24%) compared to the untreated unirradiated control, while the treated irradiated control was not significantly thicker than the unexposed control. Skin sensitivity was tested using a modification of the technique of esthesiometry, by observing rapid avoidance responses of the mouse to a pressure of 0.96 g/cm2 exerted by applying to the skin the tip of a nylon esthesiometer fiber extended to 60 mm in length. The untreated irradiated mice were more sensitive (p less than 0.07, Wilcoxon test) than the treated irradiated mice, and also significantly different from the untreated unirradiated control mice (p less than 0.04, Wilcoxon test), but the treated irradiated mice were not significantly differently sensitive when compared to the unirradiated controls (p less than 0.32). Taken together these data indicate that the erythema, edema, and skin sensitivity commonly associated with UVB-induced sunburn are significantly reduced by topical application of tocopherol acetate even after the exposure has occurred. This observation suggests that treatment of sunburn may be possible even after the irradiation has stopped, by a derivative of d-alpha-tocopherol which is stable to autooxidation.

A possible role for vitamins C and E in cataract prevention.

Biochemical evidence suggests that oxidative stress caused by accumulation of free radicals is involved in the pathogenesis of senile cataracts. If so, appropriate amounts of the antioxidant vitamins C and E might be expected to prevent or retard the process. Such activity has been observed in several in vitro and in vivo studies of experimentally-induced cataracts. A recent epidemiologic study found that cataract patients tended to have lower serum levels of vitamins C, E, or carotenoids than did control subjects. The present investigation, which compared the self-reported consumption of supplementary vitamins by 175 cataract patients with that of 175 individually matched, cataract-free subjects, revealed that the latter group used significantly more supplementary vitamins C and E (P = 0.01 and 0.004, respectively). Because the results suggested a reduction in the risk of cataracts of at least 50%, a randomized, controlled trial of vitamin supplementation in cataract prevention may be warranted.

Differentiation of rat lens epithelial cells in tissue culture. II. Effects of cytochalasins B and D on actin organization and differentiation.

Abstract Cytochalasins B and D were used to investigate the involvement of microfilaments in the differentiation of rat lens epithelial cells in tissue culture. Two questions were asked: (1) Does the organization of microfilaments change upon morphological differentiation of the lens epithelial cell? (2) Is the change in the organization of microfilaments required for the production of the differentiation-specific protein, γ-crystallin? Cytochalasin B arborized differentiating lens epithelial cells and had no effect on the undifferentiated cells. Immunofluorescent staining of these two types of cells revealed significant differences in the organization of actin. Actin appeared as longitudinal filaments in the differentiating cells, while it appeared in a diffuse nonfibrillar form in the undifferentiated cells. This indicated changes in the organization of actin during differentiation. Cytochalasin B caused a decline in cell number at 10−6–10−5 M. However, only that concentration which caused arborization of cells and disruption of microfilaments (10−5 M) inhibited morphological differentiation and production of γ-crystallin. Cytochalasin D (10−7–10−5 M) did not cause a dramatic decrease in cell number; nevertheless, it induced the arborization of cells and disruption of microfilaments at lower concentrations (10−7–10−6 M) and inhibited morphological differentiation and production of γ-crystallin at lower concentrations (10−7–10−6 M) than did cytochalasin B. Thus, only those concentrations of cytochalasins which disrupt microfilaments and prevent their organization into filamentous form seem to inhibit differentiation. This suggests that the organization of actin is required for the program of differentiation of the lens epithelial cells.

[55] High-performance liquid chromatography-electrochemical detection of antioxidants in vertebrate lens: Glutathione, tocopherol, and ascorbate

The HPLC-EC method has good specificity for the analysis of glutathione, tocopherol, and ascorbate. The same HPLC system can be used for all three analysis with changes of mobile phase and the electrode cell to match the procedure required. The same C18 reversed-phase column has been used with a refillable guard column for 3 years with no noticeable loss of resolving power. The main advantage of the glutathione procedure was the ability to monitor both GSH and GSSG, which allowed us to confirm that loss of GSH in the diabetic rat lens does not result in the appearance of GSSG. The main benefit of the tocopherol procedure was the ability to measure the tocopherol content of a single rat lens. Our previous experience with UV or fluorescence detection showed those methods to be not sensitive enough for a single lens determination. The mammalian lens has the lowest tocopherol content of the tissues of the eye, 10 to 40 times less than most body tissues as measured by gas chromatography-mass spectrometry (GC-MS). The better sensitivity of electrochemical detection has allowed for a single lens determination, keeping the number of experimental animals to a minimum. An advantage of the ASC analysis procedure was the extra specificity imparted by both the chromatography and the detector as well as the ability to estimate the total ascorbate (ASC plus DHAA) and DHAA content. Other reducing agents such as GSH and uric acid can interfere in colorimetric methods that rely on the reducing action of ASC. The very high GSH content of the mammalian lens was a concern when choosing a procedure. GSH levels exceeding 10 times the level of lens samples were found to yield no response using the HPLC-EC procedure for ASC. The only disadvantage with electrochemical detection was that the electrode response could drift with time, requiring more frequent calibration with standards. We continue to utilize these methods to examine the prevacuole loss of ASC and GSH in the diabetic rat lens model of cataract.

Modelling cortical cataractogenesis. XI. Vitamin C reduces γ-crystallin leakage from lenses in diabetic rats

Normal and streptozotocin diabetic female Wistar rats were given vitamin C (VC) at 0.3% or 1.0% (w/w) in the diet: 1% dietary VC resulted, in 12-24 hr, in significant increases in serum ascorbate levels and lens ascorbate concentrations in normal rats. The increase was biphasic, with VC concentrations falling to a lower level which was still significantly elevated compared to controls in the period of 1.7-4 days for serum and 1.7-5 days for lenticular VC. At the end of 10 weeks the rats were examined for weight gain or loss, general body condition and cataracts. At the time of killing, blood was collected for measurement of serum glucose. Alpha-crystallin levels were determined in vitreous and aqueous humours using a radioimmunoassay. One lens from each rat was fixed for either scanning electron microscopy or light microscopy; the other lens was homogenized in 8 M guanidinium chloride for adenosine triphosphate analysis. In normal rats, a small amount of gamma-crystallin was found in the vitreous humour, and an even smaller amount in the aqueous humour. Diabetes caused a five-fold increase in the vitreous humour and a 2.5-fold increase in gamma-crystallin in the aqueous humour. Diabetes also led to a significant worsening in general body condition, loss of body weight, formation of cataracts, and decrease in lens adenosine triphosphate levels. Addition of VC to the diet of diabetic animals resulted in reduction in cataracts and a decrease of gamma-crystallin leakage into the aqueous and vitreous humours. VC had no effect on lens adenosine triphosphate levels.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro studies of microwave-induced cataract: Separation of field and heating effects

Abstract A parametric study of heat and pulsed microwave energy was performed on murine ocular lens in vitro. Adult rat lenses were placed in a specially designed chamber with thermostat in which temperature was maintained by the circulation of thermostatically regulated phosphate-buffered saline, during irradiation with 915 MHz microwaves. Irradiation in pulsed (Pu) mode was used. Lenses exposed at 37°C to pulsed irradiation at specific absorption rates (SAR) equal to or greater than 400 mW/g, and immediately fixed, showed numerous small holes (not previously reported for other in vitro cataracts) in the equatorial cell surfaces, when examined by scanning electron microscopy (SEM). At 39°C with SAR equal to or greater than 120 mW/g similar holes appeared. On continued incubation for two days the holes could not be seen as easily because globular degeneration appeared to advance into the equatorial areas where holes had originally been seen. In lenses exposed to 120 mW/g at 37°C globular degeneration and equatorial subcapsular foam were observed. At SAR values equal to or above 400 mW/g at 39°C and 1·2 W/g at 37°C, large globules characteristic of temperature elevation to 47 or 50°C were observed, indicating that the effect of the electromagnetic field itself in the absence of any comparable temperature elevation, was equivalent to a 10°C rise in temperature. An unexpected finding was the apparent fragmentational stretching of the capsule observed by SEM, in lenses fixed immediately after irradiation at 37°C at an SAR value of 1·2 W/g. These effects: holes, foam, large globules and capsular damage are apparent examples of physical damage to the lens. Modern radars using directional antennas can deliver pulse power densities many times higher than those employed in these experiments. This work indicates that additional effects of microwave irradiation dependent on modulation should also be considered as potential hazards in setting safety standards.

Eye lens opacity in cortical cataracts associated with actin-related globular degeneration.

Abstract Incubation of rat lenses with intact capsules in vitro in Medium 199 as described by Patterson and Fournier 1976 resulted in no change in clarity or morphological appearance of the lens cells during one week when examined by scanning electron microscopy. Incubation of such lenses in medium containing the specific inhibitor of microfilament function, cytochalasin D, for one week, gave rise to a cortical cataract. These lenses exhibited loss of acuity, opacity and associated globular degeneration of the lens cortex similar to that previously observed by scanning electron microscopy in human and animal cataracts. Although several mechanisms have been suggested for cytochalasin D, the most likely one, from our reading of the literature, is that it appears to act on actin microfilaments; this suggests that microfilaments may be important components of the lens cell architecture since disorganization of only one cellular element—actin microfilaments, may be sufficient to cause the opacity and globular degeneration. Study of the time course of appearance of the globular degeneration indicates that the loss of acuity and opacity occur concurrently with it, and are related to the extent of the globular degeneration. Up to 24 hr, the globular degeneration was almost completely reversible, along with the opacity. Since the effect of cytochalasin D on microfilaments is reversible, this is consistent with the opacity and globular degeneration being caused by disorganization of microfilaments by cytochalasin D. Such actin-related globular degeneration may be a contributing factor in cortical cataractogenesis.

Modelling cortical cataractogenesis: VI. Induction by glucose in vitro or in diabetic rats: Prevention and reversal by glutathione

Cataractogenesis can be induced by glucose in the rat lens in vitro and in vivo. In vitro, this is done by increasing the amount of glucose in the medium surrounding the isolated lens; within 48 hr considerable globular degeneration is seen subcapsularly, deeper in the equatorial region. In vivo, it is achieved by making the rat diabetic by injecting streptozotocin i.v., which selectively destroys the beta-cells of the pancreas; the blood serum glucose level increases markedly, and thence the aqueous humour level and, in turn, the lens concentration. Globular degeneration occurs as in vitro, but not until 6 weeks is a degree of damage observed comparable to that seen in the lenses incubated in vitro for only 48 hr. Lenticular sorbitol and fructose are also markedly elevated as a result of the high glucose levels. If glutathione (GSH) is present in the medium (0.1 mM) or injected s.c. daily into the diabetic rats, there is no evidence of subcapsular globular degeneration of the cortical fiber cells, even though the lenticular levels of glucose, sorbitol and fructose are the same as when GSH was not given; this is true for either the in vitro or in vivo situation, although individual values in the two situations do differ somewhat from one another. When rats were given GSH beginning several weeks after the diabetic state had been induced, the damage subsequently observed was much less than if the rats had been diabetic without GSH for the same total length of time; it was also much less than damage which should have occurred by the time GSH treatment was instituted. It would thus appear that a certain amount of reversal of the globular degeneration is possible, although damage in the equatorial region (wedge-shaped) seems less amenable to rescue by glutathione. The data indicate that glutathione can prevent or diminish the severity of sugar cataractogenesis, and that there would appear to be more steps in sugar cataractogenesis than simply osmotic damage, although this may be the primary event.

Modeling cortical cataractogenesis V. Steroid cataracts induced by solumedrol partially prevented by vitamin E in vitro

Rat lenses incubated in tissue culture medium (M 199) maintain their transparency for a long period of time. The soluble corticosteroid, solumedrol (methyl prednisolone sodium succinate) was added to the medium, at concentrations including the range expected during rejection episodes following organ transplantation (3.8 X 10(-9) M-3.8 X 10(-6) M). At the lowest level used (3.8 X 10(-9) M), five lenses of 12 became opaque following a 48 hr incubation, while at higher concentrations of solumedrol almost all lenses developed opacities. Addition of vitamin E to the medium resulted in partial prevention of the cataract as judged by the smaller proportion of lenses becoming opaque. Examination of the lenses by scanning and transmission electron microscopy (SEM and TEM, respectively), indicated that in untreated lenses the initial location of the cataract is at the anterior pole of the lens where a deepening area of degeneration formed, followed by a uniform subcapsular layer of degeneration spreading over the remainder of the lens. Damage at this location is not typical of most in vitro cortical cataracts. In the presence of vitamin E the extent of damage was less, involving, initially, an equatorial wedge of globular degeneration and spreading anteriorly and posteriorly in a thinner subcapsular layer. This type of damage was more typical of that seen previously for cataracts induced by cytochalasin D, elevated glucose and hygromycin B.