Inhibition of delta-aminolevulinic acid dehydratase in human red blood cells by lead and activation by zinc or cysteine.

Inhibition of blood delta-aminolevulinic acid dehydratase(ALA-D) activity by lead was studied in vivo and in vitro. In vivo, a negative linear correlation (r = -0.85) was found between the logarithmic values of ALA-D activity and blood lead levels. In vitro the inhibitory effect of lead on blood ALA-D activity increased both with contact time and contact temperature of lead with blood before ALA-D assay. Maximum enzyme inhibition occurred after 14 h of contact at 25 degrees C. Inhibition of ALA-D activity by lead, in vivo as well as in vitro, is suppressed by the addition of zinc or cysteine. The logarithmic values of the activity ratios increase linearly with blood lead concentrations. The increase in ALA-D activity brought about by the addition of zinc or cysteine can be used to identify cases of low enzyme activity with no lead intoxication involved. The same technique can also detect cases in which ALA-D inhibition may be concealed by a presumably high initial enzyme activity as observed in some patients.     

S-adenosyl-L-methionine a counter to lead intoxication?

The effect of S-adenosyl-L-methionine (SAM) administration to both acute and chronic lead exposed mice was investigated. SAM was given s.c. at different doses and for different time intervals. The best results were obtained using 20 mg SAM/kg applied daily over a period of 20-22 days. Results obtained in both acute and chronic lead poisoning were quite similar. GSH concentration in blood and liver, reduced in intoxicated animals was increased after SAM administration reaching normal values. Blood, liver and kidney lead content notably increased at the beginning of SAM treatment and decreased rapidly in the group receiving SAM, attaining values near control levels in 2 weeks. A significant recovery of blood, liver, kidney, spleen and brain delta-aminolevulic acid dehydratase (ALA-D) initially reduced in poisoned animals, was clearly produced after SAM administration. A clear and direct correlation between the recovery of both ALA-D activity and GSH levels and the decreased concentration of lead in tissues was observed, reinforcing our proposal that enhancement of thiol content as a result of SAM administration would facilitate the detoxification process and lead removal, consequently reversing the inactivation of the enzyme. We conclude that SAM therapy is beneficial in the treatment of lead intoxication.     

In inherited porphyrias, lead intoxication is a toxogenetic disorder

1. delta-Aminolevulinic acid dehydratase (ALA-D), blood lead and several enzymes and metabolites of the heme biosynthetic pathway were measured in a number of symptomatic porphyric patients, 22 with acute intermittent porphyria, three with hereditary hepatic coproporphyria, 10 with hereditary porphyria cutanea tarda, two with erythropoietic protoporphyria and two with congenital erythropoietic porphyria and in 84 lead intoxicated persons. 2. In the 39 individuals suffering from the inherited porphyrias and in 32 lead poisoned patients with a 30-50% reduced deaminase, blood lead content was not sufficiently increased (average 28 micrograms%) to account for the greatly decreased activity of ALA-D (average 36% of controls). 3. After a relatively trifling lead exposure they developed the signs of acute lead intoxication. 4. A second group of lead intoxicated patients showing low ALA-D activity and corresponding high concentration of lead in blood, exhibited no other physiologic deviation in the enzymes and metabolites of porphyrin biosynthesis. 5. Individuals with inherited porphyrias are ultrasensitive to low level lead exposure and that lead would also act as a triggering factor. In these patients, lead intoxication can be considered a toxogenetic disorder. 6. An inversely linear correlation between ALA-D activity and blood lead content was obtained for both groups of lead intoxicated patients, however, a different constant (k) for each was obtained, which we have taken as a measure of lead toxogeneticity: k = 10 +/- 1 for lead intoxicated individuals with otherwise normal heme metabolism and k = 5 +/- 0.5 for lead intoxicated symptomatic porphyric patients. 7. Determination of erythrocytic ALA-D, besides blood lead, will be a valuable indicator for preventive medical care for these patients, when they are expected to be exposed to lead either environmentally or in their professional life.     

Beneficial effect of S-adenosyl-L-methionine in lead intoxication. Another approach to clinical therapy

Five patients with chronic lead intoxication were treated with S-adenosyl-L-methionine (12 mg/kg body weight, daily), given intravenously, over a period of 22 days. A significant recovery of erythrocytic ALA-D was observed in all cases, after therapy. Blood lead content significantly pathologic at the beginning of SAM administration, rapidly decreased within 24-48 h of initiating treatment, reaching nearly control values at the end of the trial. A good correlation between recovery of ALA-D activity and decreased concentration of lead in RBC was found. GSH content in blood was diminished in lead poisoned patients, increasing to normal levels after SAM administration. Other biochemical parameters such as Deaminase activity in RBC, ALA, PBG, porphyrins and lead in urine and serum gamma-GT were measured, showing no important deviations from control values before, during or after treatment. Both biochemical and clinical improvement was observed, indicating that SAM therapy is beneficial in the treatment of lead intoxication. No untoward signs were observed. The mechanism of action of SAM is not yet clear; however, a chelating effect could be excluded, and very likely its action can be attributed to glutathione availability.     

Ginkgo biloba supplement abates lead-induced endothelial and testicular dysfunction in Wistar rats via up-regulation of Bcl-2 protein expression,pituitary-testicular hormones and down-regulation of oxido-inflammatory reactions

BackgroundApoptotic and oxido-inflammatory pathways have been found to be up-regulated in lead acetate poisoning which has been associated to endothelial and testicular dysfunctions. It is yet uncertain, nevertheless, if treatment with Ginkgo biloba supplements (GBS), a flavonoid-rich natural product can lessen the adverse effects of lead on endothelial and testicular functions. This study investigated the impact of Ginkgo biloba supplementation on lead-induced endothelial and testicular dysfunctions.MethodsThe animals were treated with GBS (50 mg/kg and 100 mg/kg orally) for 14 days following oral exposure to lead acetate (25 mg/kg) for 14 days. After euthanasia, blood samples, epididymal sperm, testes, and aorta were collected. The quantities of the hormones (testosterone, follicle stimulating hormone (FSH) and luteinizing hormone (LH), as well as the anti-apoptotic, oxidative, nitrergic, inflammatory markers, were then determined using immunohistochemistry, ELISA, and conventional biochemical methods.ResultsGBS reduced lead-induced oxidative stress by increasing the levels of the antioxidant enzymes catalase (CAT), glutathione (GSH), and superoxide dismutase (SOD), while lowering malondialdehyde (MDA) in endothelium and testicular cells. Normal testicular weight was restored by GBS which also decreased endothelial endothelin-I and increased nitrite levels. TNF-α and IL-6 were decreased while Bcl-2 protein expression was enhanced. Lead-induced alterations in reproductive hormones (FSH, LH, and testosterone) were also restored to normal.ConclusionAccording to our result, using Ginkgo biloba supplement prevented lead from causing endothelial and testicular dysfunction by raising pituitary-testicular hormone levels, boosting Bcl-2 protein expression and lowering oxidative and inflammatory stress in the endothelium and testes.     

delta-Aminolevulinic acid dehydratase activity in erythrocytes of diabetic patients

Erythrocytes delta-aminolevulinate dehydratase activity was assayed in 41 diabetic patients and 33 normal controls. It was found that in diabetic patients the erythrocyte ALA-D activity was lower than in controls, and the difference of the mean values was statistically highly significant (P less than 0.001). We found a significant negative correlation (r = - 0.846, P less than 0.001) between ALA-D activity and blood glucose levels. For this reason, using normal adult human whole blood haemolysates, it was investigated the effects in vitro of glucose and insulin on normal erythrocytic ALA-D. No significant difference in ALA-D activity was found in the presence of insulin. On the other hand, there was considerable decrease in the enzyme activity in the blood samples after glucose addition.     

Biochemical changes in longear sunfish, Lepomis megalotis, associated with lead, cadmium and zinc from mine tailings

Longear sunfish were collected from a stream contaminated with mine tailings rich in lead (Pb), cadmium (Cd) and zinc (Zn). Blood samples were analysed for δ-aminolevulinic acid dehydratase (ALA-D) activity and Pb concentration. Vertebrae were tested for bone strength and composition, and Pb, Zn, and Cd concentrations were determined in muscle tissue. ALA-D activity was negatively correlated with blood Pb concentration ( r =–0.66), and enzyme activity was significantly higher and blood Pb significantly lower at the reference site than at the contaminated sites. Blood Pb was highly correlated with Pb in muscle tissue ( r = 0.72), and the concentrations of Pb and Cd in muscle tissues were themselves correlated ( r = 0.64). In fish from contaminated sites, two of the mechanical properties of the vertebrae measured (elastic limit and modulus of elasticity) were significantly different from values in fish from the reference site. These properties and one other (stress) were weakly correlated with muscle Cd concentration (0.42 < r < 0.46). Biochemical differences among fish from different sites were also evident; concentrations of calcium, phosphorus and collagen were lower in the vertebrae of fish from some of the contaminated sites than at the reference site, and bone phosphorus was negatively correlated with concentrations of Pb in both muscle ( r =– 0.62) and blood ( r =– 0.75). Collectively, these results indicate that, in addition to the well-documented effects of Pb on haem synthesis, other important biochemical pathways may be disrupted by continuous low-level exposure to elemental contaminants.     

The effect of exogenous oxytocin on streptozotocin (STZ)-induced diabetic adult rat testes

Oxytocin (OXY) plays a crucial role in reproduction. The aim of this study is to investigate the therapeutic and protective effects of oxytocin treatment on streptozotocin (STZ) induced diabetes in testicular tissue. The rats were randomly divided into four experimental groups: (I) Control Group, (II) STZ induced Diabetic Group (STZ Group), (III) STZ induced Diabetic Group with Pre-Oxytocin treatment (Pre-OXY Group) and (IV) STZ induced Diabetic Group with Post-Oxytocin treatment (Post-OXY Group); each group contains six animals. The rats whose blood glucose levels were more than 200 mg/dl were included to the experiment. At the end of the 4th week, testes tissue samples were taken to be processed for light microscopy and transmission electron microscopy. Malondialdehyde (MDA), Glutathione (GSH) and Advanced Oxidation Protein Products (AOPP) levels were determined biochemically in blood samples. Testicular tissue samples stained with Hematoxylin and Eosin (H&E) and Periodic acid-Schiff (PAS) reaction were evaluated under light microscope. The histopathological damage score of testicular tissue, which was significantly increased in STZ group, was decreased by oxytocin treatment. According to biochemical data, MDA and AOPP levels have been increased in the blood of STZ Group compared to the Control Group whereas they decreased significantly in Oxytocin-treated Groups compared to STZ Group. GSH levels were significantly decreased in the blood of STZ Group and increased in the blood of Oxytocin-treated Groups compared to STZ Group. In conclusion, oxytocin has a potential protective effect on the testes tissue of STZ-induced diabetic rats.     

The effect of aluminum chloride on some steps of heme biosynthesis in rats after oral exposure

Certain disturbances in heme biosynthesis induced by aluminum chloride were examined. The experiment was performed on female rats that received AlCl₃ orally at the dose 100 mg Al/kg daily for 21 d. The effects of aluminum on the activity of delta-aminolevulinic acid synthetase (ALA-S), dehydratase (ALA-D), and heme oxygenase (O.H.) were observed on 3, 7, 14, and 21 d in liver and kidneys of rats. Also the activity of ALA-D in blood and the concentration of delta-aminolevulinic acid (ALA-U) in urine were observed. Orally administered aluminum caused increase in the activity of ALA-D in the liver and blood, and parallel decrease of ALA-U in urine (r=−0.85) of rats. Aluminum chloride also induced an increase of ALA-S and O.H. in the liver but not in the kidneys. The changes of the enzymes activity participating in heme biosynthesis after administration of aluminum may be correlated with anemia and iron metabolism in rats.     

Simvastatin Treatment Ameliorates Injury of Rat Testes Induced by Cadmium Toxicity

Cadmium-induced testicular toxicity is mediated through oxidative stress and inflammation which eventually lead to cell death. Simvastatin, the antihyperlipidemic agent, exhibits additional antioxidant and anti-inflammatory activities. The aim of the present work was to investigate the protective effect of simvastatin against cadmium-induced testicular toxicity in rats. The rats received a single intraperitoneal (i.p.) injection of cadmium chloride (2 mg/kg). Simvastatin treatment (5 mg/kg/day, i.p.) was applied for three consecutive days, starting 1 day before cadmium administration. Cadmium significantly decreased serum testosterone, and testicular reduced glutathione and catalase activity, and significantly increased testicular malondialdehyde, nitric oxide, and cadmium ion levels. Simvastatin significantly ameliorated the biochemical changes induced by cadmium. Cadmium-induced testicular tissue injury observed by histopathological examination was attenuated by simvastatin. In addition, simvastatin significantly decreased the expression of inducible nitric oxide synthase, cyclooxygenase-2, tumor necrosis factor-α, nuclear factor-κB, and caspase-3, and increased heme oxygenase-1 expression in testicular tissue of rats exposed to cadmium toxicity. It was concluded that simvastatin, through its antioxidant and anti-inflammatory activities, provided a significant protective effect against cadmium-induced testicular toxicity in rats. However, starting treatment with simvastatin before cadmium exposure, as done in the present work, is not clinically applicable. Therefore, other investigations are needed to assess the protective effect of simvastatin treatment following induction of cadmium testicular toxicity.     

Inherited deficiency of delta-aminolevulinic acid dehydratase.

Delta-aminolevulinic acid dehydratase (ALA-D) is the second enzyme in the porphyrin-heme pathway and converts delta-aminolevulinc acid (ALA) to porphobilinogen (PBG). A family is reported with an inherited deficiency of red cell ALA-D activity occurring over three generations in an autosomal dominant pattern. Intial experiments support the hypothesis that the mutation in this family may affect a regulatory gene, but enzyme purification and further study are required. Although no clinical manifestations of deficient ALA-D activity have been found in affected persons, families such as this may be at increased risk for the serious consequences of lead poisoning, which produces marked inhibition of ALA-D activity.     

Distribution of delta-aminolevulinic acid synthetase and delta-aminolevulinic acid dehydratase in liver and kidney of rainbow trout (Salmo gairdnerii)

1. Activities of delta-aminolevulinic acid synthetase (ALA-S) and delta-aminolevulinic acid dehydratase (ALA-D) in trout liver and kidney were compared with those in the mouse. 2. ALA-S activity (per unit tissue fresh weight) exceeded ALA-D activity in trout liver and kidney. 3. In trout kidney, ALA-S activity slightly exceeded, and ALA-D activity far exceeded, their activities in trout liver. 4. In trout, heme synthesis differs from that in mammals in that appreciable synthesis occurs in the kidney, and in that ALA-S activity is not rate limiting.     

Naringenin alleviate reproductive toxicity evoked by lead acetate via attenuation of sperm profile and biochemical alterations in male Wistar rat: Involvement of TGFβ/AKT/mTOR pathway

Exposure to Lead -causes testicular dysfunction through oxidative stress, inflammation, and apoptosis; however, naringenin (NGN) therapeutic impact against lead-evoked testicular dysfunction remains elusive. Herein, the point of the study was to examine the defensive impact of NGN on testicular dysfunction initiated by lead. Seventy-Two male Wistar rats were allotted into nine groups; control group, drug control groups, lead acetate group, as well as NGN treated groups (10, 25, and 50 mg/kg) respectively, given 5 days before lead acetate treatment. The result showed clearly the impact of lead on reduced sperm count, sperm motility as well as serum testosterone and LH levels. Additionally, it caused a significant rise in testicular inflammatory markers TNF-α, IL-1β, and TGFβ, effects that were accompanied by a reduction of AKT and mTOR levels. Lead acetate also caused degenerative changes in the testis, atrophy, and loss of spermatogenic series. Our findings revealed that NGN in a dose-dependent manner improved spermiotoxicity induced by lead acetate via restoration of the testicular function, preservation of spermatogenesis, halting inflammatory cytokines along with the enhancement of germ cell survival using upregulation of AKT/mTOR expressions. The present study discloses that NGN suppresses lead acetate toxicity that is involved in the antioxidant effect in a dose-dependent manner, besides its anti-inflammatory property.     

Regulatory role of δ-aminolevulic acid dehydratase in the dimorphic fungus Mucor rouxii

• 1.1. With the aim of finding a possible relationship between the known dimorphism phenomenon existing in the fungus Mucor rouxii and the biosynthesis of respiratory pigments, the activity of aminolevulic acid synthetase (ALA-S) and ALA-dehydratase (ALA-D) was studied in crude extracts and in 15,000 g supernatants of both mycelium and yeast-like cells.
• 2.2. The activity of ALA-S was unusually high (3 nmol ALA/hr/mg protein) compared with that reported for other tissues and did not vary with the fungus morphology.
• 3.3. Instead, ALA-D specific activity was found to be 16.5 nmol PBG/hr/mg protein in mycelium extracts, that is 7-fold greater than that measured in the yeast-like morphology (2.6 nmol PBG/hr/mg protein).
• 4.4. It was of importance to determine the activity levels of ALA-D along with the morphogenic transition from yeast to mycelium. It was observed that the greatest change and enhancing of specific activity occurred 2 hr before the emergence of the germ tubes and was held constant up to the complete development of mycelium.
• 5.5. Both hyphae formation and enhancement of ALA-D activity were diminished when cAMP was added to the culture shifted from the anaerobic atmosphere to air.
• 6.6. These findings and preliminary studies on the characterization of M. rouxii ALA-D indicate that this enzyme plays a regulatory role in porphyrin biosynthesis in this fungus as well as a key function in the characteristic morphogenic transition.

     

Testicular zinc, copper and iron concentrations in male rats exposed to subacute and subchronic formaldehyde gas inhalation.

The level of trace elements such as Zn, Cu and Fe in testicular tissue is an indication of the condition of the tissue as these elements take over important tasks. Zinc and copper are the prosthetic groups of several metalloenzymes including superoxide dismutase (SOD) which is an important antioxidant enzyme in the cellular protection from reactive oxygen species. If concentrations of these trace elements decrease significantly, SOD cannot detoxify harmful oxygen species. In this study, adult male rats (wistar-albino) were exposed to formaldehyde at different periods (subacute and subchronic) and concentrations (0; 12.2; 24.4 mg.L-1). Body and testis weights were recorded and compared with control groups. The metals described above were determined in rat testicular tissue by atomic absorption spectrometry by using wet ashing. We conclude that subacute or subchronic exposure to formaldehyde have caused growth retardation and altered levels of trace elements, including copper, zinc and iron, in testicular tissue, and may induce further oxidative damage on testicular tissue leading to spermatozoal abnormalities.     

Lead toxicity and the hypothalamic-pituitary-testicular axis

Environmental exposure to toxic levels of lead occurs in a number of industries with potential adverse effects on the reproductive capacity of exposed men. Clinical and animal studies indicate that abnormalities of spermatogenesis result from toxic lead exposure, but the pathogenetic mechanisms involved have not been identified. In order to ascertain what reproductive abnormalities occur in experimental animals when exposed to low levels of lead, 52-day-old animals were treated with water containing 0.0% (control), 0.1%, or 0.3% lead acetate for 30 days prior to killing. Whole blood serum lead levels were below detection (less than 7 micrograms/dl) in the control animals, 34 +/- 3 micrograms/dl in the 0.1% group, and 60 +/- 4 micrograms/dl in the 0.3% group (P less than 0.001). Significant negative correlations between whole blood lead levels and serum and intratesticular testosterone values were found (r = 0.64, P less than 0.001 and r = 0.6, P less than 0.001, respectively). As the level of lead exposure increased, intratesticular sperm counts significantly decreased (r = 0.81, P less than 0.001). No significant changes in serum luteinizing hormone (LH) values were found, but sperm follicle-stimulating hormone (FSH) values were significantly suppressed (P less than 0.05) after lead treatment. There was a significant decrease in ventral prostate weight (P less than 0.05), but no differences in testicular or seminal vesicle weights. Our data indicate that dietary exposure to lead resulting in whole blood serum lead values considered acceptable in the workplace (less than or equal to 40 micrograms/dl) causes inhibition of testicular function.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protection of arsenic-induced testicular oxidative stress by arjunolic acid

Arsenic-induced tissue damage is a major concern to the human population. An impaired antioxidant defense mechanism followed by oxidative stress is the major cause of arsenic-induced toxicity, which can lead to reproductive failure. The present study was carried out to investigate the preventive role of arjunolic acid, a triterpenoid saponin isolated from the bark of Terminalia arjuna, against arsenic-induced testicular damage in mice. Administration of arsenic (in the form of sodium arsenite, NaAsO(2), at a dose of 10 mg/kg body weight) for 2 days significantly decreased the intracellular antioxidant power, the activities of the antioxidant enzymes, as well as the levels of cellular metabolites. In addition, arsenic intoxication enhanced testicular arsenic content, lipid peroxidation, protein carbonylation and the level of glutathione disulfide (GSSG). Exposure to arsenic also caused significant degeneration of the seminiferous tubules with necrosis and defoliation of spermatocytes. Pretreatment with arjunolic acid at a dose of 20 mg/kg body weight for 4 days could prevent the arsenic-induced testicular oxidative stress and injury to the histological structures of the testes. Arjunolic acid had free radical scavenging activity in a cell-free system and antioxidant power in vivo. In summary, the results suggest that the chemopreventive role of arjunolic acid against arsenic-induced testicular toxicity may be due to its intrinsic antioxidant property.     

Effects of boric acid feeding on the oxidative stress parameters in testes,sperm parameters and DNA damage in mice

This study was aimed to determine the effects of boric acid on oxidative stress, testicular tissue and spermatozoon DNA. Experiments were performed with Swiss Albino mice divided equally into two groups based on the tratment period: one for 4 and the other for 6-week duration. These groups were further divided into subgroups as Control and those administered daily at oral doses of 115 mg/kg, 250 mg/kg and 450 mg/kg of boric acid. Then, testicular tissue were examined postmortem and analyzed using ex-vivo biochemical tools for oxidative stress, spermatozoon membrane integrity, sperm motility and live cell rate (%). In both 4 and 6-week groups, v. seminalis weight, membrane integrity, motility, live cells and GSH levels exhibited a decreasing trent compared to the controls. In addition, 6-week group had a decrease in SOD level. MDA level was higher in controls in both 4 and 6-week groups. Spermatozoon DNA was intact in the 4-week group, but damaged in the 6-week group, and the degree of the damage dependent on the administered dose. Boric acid induces oxidative stress in testicular tissue, and its long-term application (only 6 weeks) caused damage in spermatozoon DNA.     

Disturbances in heme biosynthesis in rabbits after administrationper os of low doses of tin or lead

The experiment was performed on female rabbits that receivedper os equimolar doses (17 μM Me/kg) of SnCl₂×2H₂O or Pb (CH₃ COO)₂ every day for 5 d. The activity of δ-aminolevulinic acid dehydratase (ALA-D) in the whole blood, liver, kidneys, brain, spleen, and bone marrow, concentration of free erythrocyte protoporphyrins (FEP), activity of δ-aminolevulinic acid synthetase (ALA-S) in the liver and bone marrow, urine δ-aminolevulinic acid (ALA-U), and coproporphyrins (CP-U) were determined. Lead and tin concentrations in the blood were estimated. Lead caused a significant inhibition of ALA-D in the blood, increased FEP concentration, and ALA and CP excretion in urine of rabbits. Lead also decreased ALA-D activity in the bone marrow and in the liver, and did not change ALA-2 activity in the liver and bone marrow. Tin did not change any of the examined indices. Tin doses applied in the present study, maintained within the limits of permissible standards of metal levels in human diet, did not affect the process of heme biosynthesis in rabbits.     

Effect of chronic vanadium administration in drinking water to rats

Two-month old Wistar rats of both sexes received, as sole drinking liquid, an aqueous solution of ammonium metavanadate (AMV) at a concentration of 0.01 or 0.05 mg V cm^–3 (0.2 or 1.0 mM) for a period of 4 weeks. It was calculated that the animals took up doses of 1.5 and 5–6 mg V kg body weight^–1 24 h^–1, respectively. Food and AMV solution consumption in the experimental groups was similar to food and water consumption in the control group. A statistically significant decrease of consumption of AMV solution at a concentration of 0.05 mg V cm^–3 was noted only in males. Hematological examination demonstrated a decrease in the erythrocyte count, hemoglobin level and hematocrit index. This decrease in the erythrocyte count was associated with an increased percentage of reticulocytes in the peripheral blood of the animals drinking the solution with a higher vanadium content. Biochemical analyses demonstrated a decrease of l-ascorbic acid levels in the plasma and erythrocytes of animals drinking the AMV solutions. A distinct tendency for the malonyldialdehyde level to increase in the blood was also observed. Among the enzymes examined in the erythrocytes (catalase, glucose-6-phosphate dehydrogenase, lactate dehydrogenase and -aminolevulinic acid dehydratase [ALA-D]) only ALA-D activity was depressed.