Influence of neonatal steroid (diethylstilbestrol, allylestrenol) treatment on the sexual behaviour of the adult rat

A single neonatal treatment with diethylstilbestrol (DES) or allylestrenol (AE) considerably depressed the sexual activity of male rats in adulthood. DES had a stronger depressive effect than AE. Though the adult sexual activity of intact female rats was also reduced by DES it was not influenced by AE. Ovariectomized females that had been hormone-treated before experimental mating showed reduced sexual activity under the influence of neonatal DES-treatment but increased sexual activity when treated neonatally with AE.     

Estrogen regulation of progestin biosynthetic enzymes in cultured rat granulosa cells

The mechanism by which estrogens enhance gonadotropin-stimulated ovarian progestin production was investigated by studying the modulation of pregnenolone biosynthesis as well as the activities of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) in cultured rat granulosa cells. Cells from immature hypophysectomized, estrogen-treated rats were cultured for 3 days with follicle-stimulating hormone (FSH) and/or estrogens. Pregnenolone production was measured in the presence of cyanoketone which inhibits 3 beta-HSD activity. Activities of 3 beta-HSD and 20 alpha-HSD were determined in cell homogenates by direct enzyme assays. Some cells were also primed with FSH to induce luteinizing hormone (LH) receptors for studies on the effects of estrogens on LH-modulated parameters. Pregnenolone production by cultured granulosa cells was stimulated by FSH, while treatment with diethylstilbestrol (DES) or estradiol further enhanced the gonadotropin action. Treatment with FSH increased 3 beta-HSD activity. Similarly, concomitant treatment with DES further enhanced 3 beta-HSD activity in a dose-dependent manner with an apparent ED50 of 10(-8) M. Also, treatment with estrogens alone increased 3 beta-HSD activity. The increases in enzyme activity induced by estrogen alone or in combination with FSH were not associated with changes in the apparent Km values. FSH also stimulated 20 alpha-HSD activity by 2-fold in these cells, while concomitant treatment with DES did not affect the FSH action. In FSH-primed cells, LH stimulated pregnenolone production while the LH action was enhanced by concomitant treatment with the estrogens. Likewise, LH stimulated the activity of 3 beta-HSD, while concomitant DES treatment further augmented LH action. LH did not stimulate 20 alpha-HSD activity when added alone or in combination with DES. Thus, the estrogen enhancement of the gonadotropin-stimulated progesterone production in cultured rat granulosa cells is associated with increases in pregnenolone biosynthesis and the activity of the 3 beta-HSD enzyme, without affecting the 20 alpha-HSD activity.     

Ultracytochemical localization of estrogen-stimulated guanylate cyclase in rat uterus

Estrogens are known to increase cyclic guanosine monophosphate (cGMP) levels in the uterus of rats by enhancing guanylate cyclase (GC) activity. In the present study, the cytochemical localization of GC activity was studied in the uteri of immature and ovariectomized rats after treatment with diethylstilbestrol (DES), progesterone, estrogen antagonist (CI628), and a combination of DES and CI628. Twenty-four hours after the first dose of DES, moderate to strong guanylate cyclase activity was indicated by lead phosphate precipitate on the luminal microvillar and basolateral surfaces of epithelial cells, whereas strong activity was found on the plasma membranes of fibroblasts, endothelial cells, and myometrial cells. The enzyme activity in the epithelial cells declined slightly 24 hr after the second daily dose of DES. Uterine tissues from DES-treated rats that were preheated at 60 degrees C for 30 min or preincubated with a GC inhibitor showed no reaction product. Guanylate cyclase activity was not observed cytochemically in the uterine tissues of the vehicle control (immature or ovariectomized) or progesterone-and CI628-treated animals. Weak guanylate cyclase activity was observed on the plasma membranes of epithelial cells and endothelial cells after doses of DES and CI628 were given simultaneously. The biochemical assays of the total homogenate in vitro indicated that uterine GC showed about a twofold increase after one dose of DES and a 1.3-fold increase following two doses (one dose per day) of DES when compared with their respective nontreated controls, or with progesterone-treated uteri. GC was found in particulate (09%) and cytosol (10%) fractions.These data demonstrated that DES stimulated uterine guanylate cyclase activity, while progesterone and CI628 were ineffective at the doses used. Estrogen antagonist CI628 doses not completely suppress the effect of DES.     

Decrease of hepatic catalase level by treatment with diallyl sulfide and garlic homogenates in rats and mice

Diallyl sulfide (DAS) is a flavor compound derived from garlic and is active in the inhibition of chemically induced cytotoxicity and carcinogenicity in animal models. This study was conducted to examine the effects of the treatment of DAS and garlic homogenates on the activities of catalase, glutathione peroxidase, and superoxide dismutase. Male Sprague-Dawley rats were treated with DAS i.g. at daily doses of 50 or 200 mg/kg for 8 days, causing the hepatic catalase activity to decrease by 55 and 95%, respectively. Such a decrease in hepatic catalase activity was also observed when the DAS treatment was extended to 29 days. Western blot analysis showed that the DAS treatments resulted in corresponding decreases in the liver catalase protein level. No significant change in the catalase activity in the kidney, lung, and brain was observed with the treatments, but a slight decrease in heart catalase activity was observed. These treatments did not cause significant changes in superoxide dismutase and glutathione peroxidase activities in these tissues. Treatment with DAS at a daily dose of 200 mg/kg for 1-7 days resulted in a gradual decrease in the liver catalase activity to 5% of the control level, but it did not decrease the erythrocyte catalase activity. Treatment of rats with fresh garlic homogenates (2 or 4 g/kg, i.g., daily for 7 days) caused a 35% decrease in liver catalase activity. A/J mice treated with DAS and garlic homogenates also showed a decrease in the liver catalase activity. Diallyl sulfone (DASO2), a DAS metabolite, however, did not effectively decrease catalase activity in mice. The catalase activity was not inhibited by either DAS or DASO2 in vitro. The present results demonstrate that treatment with DAS and garlic homogenates decrease the hepatic catalase level in rats and mice.     

Myristic acid increases the activity of dihydroceramide Delta4-desaturase 1 through its N-terminal myristoylation

Dihydroceramide Delta4-desaturase (DES) catalyzes the desaturation of dihydroceramide into ceramide. In mammals, two gene isoforms named DES1 and DES2 have recently been identified. The regulation of these enzymes is still poorly understood. This study was designed to examine the possible N-myristoylation of DES1 and DES2 and the effect of this co-translational modification on dihydroceramide Delta4-desaturase activity. N-MyristoylTransferases (NMT) catalyze indeed the formation of a covalent linkage between myristoyl-CoA and the N-terminal glycine of candidate proteins, as found in the sequence of DES proteins. The expression of both rat DES in COS-7 cells evidenced first that DES1 but not DES2 was associated with an increased dihydroceramide Delta4-desaturase activity. Then, we showed that recombinant DES1 was myristoylated in vivo when expressed in COS-7 cells. In addition, in vitro myristoylation assay with a peptide substrate corresponding to the N-terminal sequence of the protein confirmed that NMT1 has a high affinity for DES1 myristoylation motif (apparent K(m)=3.92 microM). Compared to an unmyristoylable mutant form of DES1 (Gly replaced by an Ala), the dihydroceramide Delta4-desaturase activity of the myristoylable DES1-Gly was reproducibly and significantly higher. Finally, the activity of wild-type DES1 was also linearly increased in the presence of increased concentrations of myristic acid incubated with the cells. These results demonstrate that DES1 is a newly discovered myristoylated protein. This N-terminal modification has a great impact on dihydroceramide Delta4-desaturase activity. These results suggest therefore that myristic acid may play an important role in the biosynthesis of ceramide and in sphingolipid metabolism.     

Hormonal regulation of activities of 4-ene-5 beta and 5 alpha-reductases and 17 beta-ol-dehydrogenase in immature golden hamster ovary

Diethylstilbestrol (DES) pellets were implanted in female golden hamsters on day 22 after birth. Hamsters with or without the DES pellet were hypophysectomized on day 23. Starting from day 26, the hypophysectomized hamsters were injected daily with 2.3-40 micrograms NIH-LH-S19, 6 or 18 micrograms NIAMD-oFSH-13, 50 micrograms NIAMD-Rat-FSH-B-1, or saline for 3 days. Ovarian homogenates from these hamsters on day 29 were incubated with [14C]-4-androstene-3,17-dione and enzyme activity (nmol/g/h) was estimated. The 5 alpha- and 5 beta-reductase activities decreased significantly following hypophysectomy. In the hypophysectomized hamster ovary, a distinct response to LH but not to FSH or DES in the 5 alpha-reductase activity was found. On the other hand, the 17 beta-ol-dehydrogenase activity was stimulated by FSH but not by LH or DES. The 5 beta-reductase activity was stimulated by DES, FSH or 2.3 micrograms LH but not by 7-40 micrograms LH. In the DES-treated, hypophysectomized hamster ovary, LH and FSH stimulated the 5 alpha-reductase and 17 beta-ol-dehydrogenase activities, respectively, but FSH or LH treatment had no significant effect on the 5 beta-reductase activity. These results show that the 5 alpha-reductase activity is regulated by LH, while the 17 beta-ol-dehydrogenase activity is stimulated by FSH in immature golden hamster ovary. The 5 beta-reductase activity seems to be regulated predominantly by FSH but the effect of FSH is largely mediated by estrogen.     

Mechanism of estrogen-induced 17-beta-hydroxysteroid dehydrogenase in ovariectomized rat uterus

Estradiol (E2), progesterone or medroxyprogesterone acetate can induce biosynthesis of the 17-beta-hydroxysteroid dehydrogenase (17-beta-HSD) in the mammalian uterus. For further understanding the 17-beta-HSD induction which may be mediated by the conjugation of the E2 to its receptor, premature ovariectomized rats were treated with E2, or with a synthetic steroid, diethylstilbestrol (DES), an agonist for the E2 receptor but not a substrate for 17-beta-HSD. Histological observation and uterus weight were examined as parameters to evaluate uterine response to those hormones at different durations of treatment. The 17-beta-HSD in ovariectomized rat uterus of each group was also examined by histochemical and biochemical assays. The results showed that the 17-beta-HSD activity in the uterus can be induced by E2 or DES, after daily treatment for 1, 14 and 28 days, but much higher in DES treated animals. The uterus weight demonstrated a "negative linear correlation" to the enzyme activity in all E2 treated groups, but not in DES or control rats. Accordingly, it was indicated that the 17-beta-HSD induction was regulated by conjugation of E2 or DES to its receptor. Therefore, we believe that the 17-beta-HSD gene in the rat uterus is another estrogen responsive gene.     

Is the cytosolic catalase induced by peroxisome proliferators in mouse liver on its way to the peroxisomes?

Dietary treatment of male C57B1/6 mice with clofibrate, nafenopin or WY-14.643 resulted in a modest (at most 2-fold) increase in the total catalase activity in the whole homogenate and mitochondrial fraction prepared from the livers of these animals. On the other hand, the catalase activity recovered in the cytosolic fraction was increased 12- to 18-fold, i.e. 30-35% of the total catalase activity in the hepatic homogenate was present in the high-speed supernatant fraction after treatment with these peroxisome proliferators. A study of the time course of the changes in peroxisomal and cytosolic catalase activities demonstrated that the peroxisomal activity both increased upon initiation of exposure and decreased after termination of treatment several days after the increase and decrease, respectively, in the corresponding cytosolic activity. This finding suggests that the cytosolic catalase may be on its way to incorporation into peroxisomes.     

Altered activation/detoxication enzymology following neonatal diethylstilbestrol treatment

The effects of neonatal exposure to diethylstilbestrol (DES) on hepatic activation/detoxication enzyme levels in the adult rat were investigated. Neonatal exposure of male rats to DES (DES males) decreased the endogenous levels of UDP-glucuronyltransferase as compared to control males. Female rats exposed neonatally to DES (DES females) had higher endogenous epoxide hydrolase and glutathione transferase activity levels than control females. Adult animals treated neonatally with DES also had altered metabolic potential following exposure to 3-methylcholanthrene and phenobarbital. The DES males treated in adulthood with 3-methylcholanthrene had higher benzo(a)pyrene hydroxylase activities and lower UDP-glucuronyltransferase activity levels than did control males treated in adulthood with 3-methylcholanthrene. The DES males exposed in adulthood to phenobarbital had reduced cytochrome P-450 and glutathione transferase activity levels as compared with respective controls. The DES females treated in adulthood with 3-methylcholanthrene had lower benzo(a)pyrene hydroxylase and epoxide hydrolase activity levels than control females receiving 3-methylcholanthrene. The DES females challenged in adulthood with phenobarbital also had decreased benzo(a)pyrene hydroxylase, epoxide hydrolase, UDP- glucuronyltransferase, and glutathione transferase activity levels as compared with respective controls. Our results demonstrated that neonatal exposure to DES changed the endogenous levels of specific hepatic enzymes and altered the metabolic response of these adult animals to a carcinogen and a drug.     

Effect of light and benzyladenine on dark-treated growing rice leaves (Oryza sativa). I. Changes in chlorophyll content and catalase activity

Light- and benzyladenine-induced reversal of the changes in chlorophyll content and catalase activity were studied in the attached first leaf of Oryza sativa L. cv. Bala, kept in darkness for different periods before maturation. Dark treatment caused a decrease in chlorophyll content and catalase activity at all times. Light treatment of dark-incubated seedlings at different periods before maturation reversed the dark-induced effect on chlorophyll content, catalase activity and dry weight and also caused a further rise in chlorophyll content compared to initial values. In darkness, the application of benzyladenine replaced the light effect in maintaining catalase activity. Chlorophyll content was also maintained by initially applied benzyladenine. Benzyladenine did not promote the photoinduced maintenance and increase in chlorophyll content and catalase activity at any time. Treatment with hydrogen peroxide, glycolate and amizol resulted in an accelerated chlorophyll breakdown and had varied effects on catalase levels. Chlorophyll decrease due to peroxide accumulation was to some extent reversible by benzyladenine, but the hormone had no effect on the peroxide-induced decrease in catalase activity. Development of catalase is light dependent. Benzyladenine stabilises the enzyme but has no effect on its synthesis.     

Diethylstilbestrol. A novel F0-directed probe of the mitochondrial proton ATPase

At low concentrations, diethylstilbestrol (DES) is shown to be a potent F0-directed inhibitor of the F0F1-ATPase of rat liver mitochondria. In analogy to other F0-directed inhibitors, DES inhibits both the ATPase and ATP-dependent proton-translocation activities of the purified and membrane bound enzyme. When added at low concentrations with dicyclohexylcarbodiimide (DCCD), a covalent inhibitor, DES acts synergistically to inhibit ATPase activity of the complex. At higher concentrations, DES restores DCCD-inhibited ATPase activity. However, there is no restoration of ATP-dependent proton translocation. Under these conditions DCCD remains covalently bound to the F0F1-ATPase complex and F1 remains bound to Fo. Significantly, when the F0F1-ATPase is inhibited by the Fo-directed inhibitor venturicidin rather than DCCD, DES is also able to restore ATPase activity. In contrast, DES is unable to restore ATPase activity to F0F1 preparations inhibited by the Fo-directed inhibitors oligomycin or tricyclohexyltin. However, combinations of [DES + DCCD] or [DES + venturicidin] can restore ATPase activity to F0F1 preparations inhibited by either oligomycin or tricyclohexyltin. Results presented here indicate that the F0 moiety of the rat liver mitochondrial proton ATPase contains a distinct binding site for DES. In addition, they suggest that at saturating concentrations simultaneous occupancy of the DES binding site and sites for either DCCD or venturicidin promote "uncoupled" ATP hydrolysis.     

Temporary decrease in renal quinone reductase activity induced by chronic administration of estradiol to male Syrian hamsters. Increased superoxide formation by redox cycling of estrogen

Cytochrome P-450-mediated redox cycling between the synthetic estrogen diethylstilbestrol (DES) and diethylstilbestrol-4',4"-quinone (DES Q) has previously been demonstrated. Cytochrome P-450 reductase catalyzes the reduction of DES Q presumably via a semiquinone formed by one-electron reduction. A reducing action of NAD(P)H quinone reductase (EC 1.6.99.2) mediating two-electron reduction of DES Q has been investigated in the present work. Quinone reductase catalyzed the conversion in the presence of NADH or NADPH of DES Q to 53-65% Z-DES, a marker product of reduction. Dicumarol (15 microM), a known specific inhibitor of quinone reductase, inhibited this reduction almost completely. Using microsomes from Syrian hamster kidney, a target organ of estrogen-induced carcinogenesis, the reduction of DES Q was only partially inhibited by dicumarol. Apparent Km values of quinone reductase and cytochrome P-450 reductase were 17.25 and 11.9 microM, respectively. These data demonstrate that in hamster kidney, quinone reductase and cytochrome P-450 reductase compete for the reduction of DES Q. Microsomal 02-. radical generation was stimulated 10-fold over base levels by the addition of 100 microM DES Q. The formation of 02-. radicals was inhibited by addition of superoxide dismutase (0.2 mg/ml) or by 2'-AMP or NADP, known inhibitors of cytochrome P-450 reductase. In contrast, dicumarol enhanced microsome-mediated 02-. formation. It is concluded that cytochrome P-450 reductase in hamster kidney microsomes mediates one-electron reduction of estrogen quinones to free radicals (semiquinones), which may subsequently enter redox cycling with molecular oxygen to form 02-.. Moreover, quinone reductase reduces DES Q directly to E- and Z-DES, and thus may prevent the formation of toxic intermediates during redox cycling of estrogens. Measurements of quinone reductase activity in liver and kidney of hamsters treated with estrogen for various lengths of time revealed a temporary decrease in activity by 80% specifically in the kidney after 1 month of chronic treatment with estradiol. Thus, a temporary decrease in quinone reductase activity, which occurred specifically in estrogen-exposed hamster kidney, may enhance the formation of free radical intermediates generated during biotransformation of estrogens.     

Effect of a single neonatal treatment with steroid hormone or steroid-like molecules on myocardial ouabain binding in the adult rat

A single neonatal treatment of rats with vitamin D3, gibberellin, allylestrenol or diethylstilbestrol (DES) influenced the ouabain binding capacity of myocardial Na, K-dependent ATP-ase. Of the active molecules tested, vitamin D3, DES and gibberellin had appreciable impact on myocardial ouabain receptors, enhancing and depressing their activity, respectively. The thymic dexamethasone and uterine estrogen receptors did not alter their binding capacity in response to neonatal exposure to vitamin D3 or gibberellin.     

Effects of postnatal DES treatment on uterine growth, development, and estrogen receptor levels

The neonatal rodent appears to be an appropriate animal model for estrogen toxicity in the developing reproductive tract. Newborn rats were treated with diethylstilbestrol (DES) at human therapeutic doses (approx 1 mg/kg) during two ontogenetic periods (postnatal days 1-5 and 1-25). Treatment on days 1-5 doubled uterine wt by day 5; however, these uteri failed to grow after discontinuation of DES treatment. In contrast, uterine wt was 4-fold higher and DNA content was 2-fold higher than controls on days 10-25 with continued DES treatment. Total uterine estrogen receptor levels, depressed 60% by day 5 of DES treatment, partially recovered after discontinuation of DES treatment but remained 25% below controls on day 25. Receptor levels following DES on days 1-25 decreased to about 15% of the controls by day 15. Short-term DES treatment approximately halved uterine gland content while continued treatment almost completely inhibited gland appearance. DES effects on glands appear related to continued hypertrophy of the luminal epithelium, from which uterine glands are derived. Subsequent failure of uterine growth caused by DES treatment on days 1-5 is similar to clinical findings of hypoplastic uteri in DES-treated patients. Disruption of the normal ontogenetic patterns of estrogen receptor by DES may be involved. These data demonstrate abnormal patterns of growth, estrogen receptor levels and morphogenesis in uteri of rats treated postnatally with DES.     

Alterations in tear biochemistry associated with postanesthetic chronic dry eye syndrome

Perioperative dry eye syndrome (DES) is a common ocular complication of long-term general anesthesia. Chronic DES can lead to permanent damage to the cornea and disturbance of visual function, up to total loss of vision. Here, a relationship between the duration of general anesthesia and the risk of chronic DES in patients was demonstrated. Using an experimental model of perioperative corneal abrasions in rabbits, it was found that introduction of animals to 3-h general anesthesia resulted in clinically significant chronic damage to the cornea in 50% of cases. The development of the complication was not associated with irreversible or long-term impairment of tear secretion, but it was accompanied by a decrease in tear film stability and growth of the total protein content as well as decrease in total antioxidant activity of the tear induced by low molecular weight antioxidants. In addition, anesthesia-induced changes in activity of tear antioxidant enzymes including superoxide dismutase and enzymes providing homeostasis of reduced glutathione (glutathione peroxidase, glutathione-S-transferase, glutathione reductase) were observed. All these alterations were protracted (up to 1-2 weeks) and therefore might account for transition of the perioperative DES into the chronic form. These findings can be useful in the development of novel approaches for the prevention and treatment of chronic forms of DES in the postanesthetic period.     

Influence of insulin status on extra-mitochondrial oxygen metabolism in the rat.

The effects of alloxan diabetes and subsequent treatment with insulin on extra-mitochondrial oxygen metabolism in terms of D-amino acid oxidase (DAAO), xanthine oxidase and catalase were examined. The DAAO activity in the liver with D-alanine and D-serine decreased by 33-62% in the diabetic group while the decrease in the kidneys was 61-74%. Insulin treatment resulted in overstimulation of DAAO activity in the liver but not in the kidneys. Tissue glycogen content was lowered in the diabetic animals but was restored by insulin treatment. Tissue glycogen content and DAAO activity showed an inverse relationship. The xanthine oxidase activity in the two tissues decreased from 40-55%; the catalase activity decreased from 34-54%. Insulin treatment was unable to restore the xanthine oxidase and catalase activities in both the tissues.     

On the synthesis and degradation of the multiple forms of catalase in mouse liver: effects of aminotriazole and p-chlorophenoxyisobutric acid ethyl ester

Aspects of the synthesis and degradation of the multiple forms of catalase in mouse liver have been investigated. The kinetics of return of catalase after aminotriazole inhibition indicated a product-precursor relationship between the granular and supernatant pools of the enzyme, and electrophoretic resolution of the individual heteromorphs of catalase during this treatment served to substantiate this relationship and indicated, in addition, that aminotriazole-inhibited catalase may be partially reactivated in the cytosol. Changes in the activity of mouse liver and kidney catalases after the administration of chlorophenoxyisobutyric acid ethyl ester (CPIB) were also monitored. After an initial decline in activity, a rapid increase to an elevated steady state occurred, with an approximately threefold increase in the liver and twofold increase in the kidney. Subcellular fractionation of the livers of CPIB-treated mice showed a massive initial increase in the supernatant pool of catalase, accompanied by a steady decrease in the activity of the peroxisomal pools. Activity increased in the peroxisomal pools at later stages of treatment, but even after a new CPIB-induced steady state was achieved, the supernatant pool of catalase remained grossly elevated. Electrophoresis of the individual heteromorphs of catalase in the supernatant after CPIB treatment showed a predominance of the most cathodal migrating forms, and turnover studies demonstrated that catalase in CPIB-treated animals exhibited a substantially lowered rate of degradation by comparison with normal animals. These results have been discussed in relation to the intracellular sequestration and turnover characteristics of catalase.     

Electrophoretic Variants of Intracellular Catalase of Different Candida Species

Intracellular and extracellular catalases of different species of Candida were investigated using different culture media. All the Candida strains produced intracellular catalase, whose enzymatic activity was detected by non-denaturating polyacrylamide gradient (4-30%) gel electrophoresis. The cell extracts presented a major 230 kDa catalase band and in some strains variants of catalase with different molecular weights were detected. Candida catalase activity was not affected by heating at 50 degrees C and incubation with beta-mercaptoethanol, but treatment with sodium dodecyl sulphate inhibited or reduced enzymatic activity. Extracellular enzyme activity was not detected in any of the culture filtrate extracts tested.     

The role of catalase in hydrogen peroxide resistance in fission yeast Schizosaccharomyces pombe.

The role of catalase in hydrogen peroxide resistance in Schizosaccharomyces pombe was investigated. A catalase gene disruptant completely lacking catalase activity is more sensitive to hydrogen peroxide than the parent strain. The mutant does not acquire hydrogen peroxide resistance by osmotic stress, a treatment that induces catalase activity in the wild-type cells. The growth rate of the disruptant is not different from that of the parent strain. Additionally, transformed cells that overexpress the catalase activity are more resistant to hydrogen peroxide than wildtype cells with normal catalase activity. These results indicate that the catalase of S. pombe plays an important role in resistance to high concentrations of hydrogen peroxide but offers little in the way of protection from the hydrogen peroxide generated in small amounts under normal growth conditions.