Synthesis and in vitro biological activity of 4alpha-(2-propenyl)-5alpha-cholest-24-en-3alpha,12 alpha-diol, a 12alpha-hydroxyl analog of 4alpha-(2-propenyl)-5alpha-cholest-24-en-3alpha-ol: the latter is a potent activator of the low-density lipoprotein receptor promoter.

4alpha-(2-Propenyl)-5alpha-cholest-24-en-3alpha-ol (3) was shown recently in a Chinese hamster ovary (CHO) cell-based low-density lipoprotein receptor/luciferase (LDLR/Luc) assay to be a potent transcriptional activator of the LDL receptor promoter in the presence of 25-hydroxycholesterol. Because of the involvement of 12alpha-hydroxylation in the metabolism of cholesterol, we are interested in investigating the effect of introducing a 12alpha-hydroxyl group to 3 on the transcriptional activity of the LDL receptor promoter. Thus 4alpha-(2-propenyl)-5alpha-cholest-24-en-3alpha,12a lpha-diol (14), a 12alpha-hydroxyl analog of 3, was synthesized from deoxycholic acid via the formation of 12alpha-[[(tertbutyl)dimethylsilyl]oxy]-4alpha-( 2-propenyl)-5alpha-cholest-24-en-3-one (11). Test results show that 14 is inactive at concentrations of up to 20 microg/ml, compared to 3 with an EC30 value of 2.6 microM, in the CHO cell-based LDLR/Luc assay. Apparently introduction of a 12alpha-hydroxyl group abolishes the capability of 3alpha-sterol 14 to activate the transcription of the LDL receptor promoter. However, in the [1-14C-acetate]cholesterol biosynthesis inhibition assay in CHO cells, 14 at 10 microg/ml (23 microM) is shown to inhibit the cholesterol biosynthesis by 51% relative to the control cells. Our previous studies indicated that 3 showed a 38% inhibition, but 4alpha-(2-propenyl)-5alpha-cholestan-3alpha-ol (1) exhibited no inhibition in the same assay at 10 microg/ml. In summary the results indicate that, in addition to the 24,25-unsaturation, the 12alpha-hydroxyl group in 14 has also conferred an inhibitory effect on cholesterol biosynthesis in CHO cells; however, the inhibition of cholesterol biosynthesis by 14 does not lead to the transcriptional activation of the LDL receptor promoter.

Synthesis and in vitro biological activity of 4alpha-(2-propenyl)-5alpha-cholest-24-en-3alpha-ol: a 24,25-dehydro analog of the hypocholesterolemic agent 4alpha-(2-propenyl)-5alpha-cholestan-3alpha-ol.

4Alpha-(2-propenyl)-5alpha-cholestan-3alpha-ol (LY295427) was previously identified from a Chinese hamster ovary (CHO) cell-based low density lipoprotein receptor/luciferase (LDLR/Luc) assay to be a potent transcriptional activator of the LDL receptor promoter in the presence of 25-hydroxycholesterol. To investigate the effect of the 24,25-unsaturation in the D-ring side chain (desmosterol D-ring side chain) on antagonizing the repressing effect of 25-hydroxycholesterol, 4alpha-(2-propenyl)-5alpha-cholest-24-en-3alpha-ol (17), a 24,25-dehydro analog of LY295427, was thus synthesized from lithocholic acid via the formation of 3alpha-[[(1,1-dimethylethyl)dimethylsilyl]oxy]-4alpha- (2-propenyl)-5alpha-cholan-24-al (15). Test results showed that 17 had an EC30 value of 2.6 microM, comparable to 2.9 microM of LY295427, in the CHO cell-based LDLR/Luc assay in the presence of 25-hydroxycholesterol. Apparently, the built-in 24,25-unsaturation in the D-ring side chain of 17 had added little effect to antagonizing the repressing effect of 25-hydroxycholesterol. In the [1-14C-acetate]cholesterol biosynthesis inhibition assay, 17 at 10 microg/ml (23 microM) has been shown to inhibit the cholesterol biosynthesis in CHO cells by 38% relative to the vehicle control; whereas LY295427 showed no inhibition in the same assay in our previous studies. In contrast to LY295427, the built-in 24,25-unsaturation in the D-ring side chain of 17 has conferred an inhibitory effect on cholesterol biosynthesis in CHO cells. In summary, the observed LDL receptor promoter activity of 17 is related to its ability to prevent 25-hydroxycholesterol from exerting the repressing effect via an undetermined mechanism and, in part, to inhibit the cholesterol biosynthesis.

Nafenopin-induced peroxisome proliferation in vitamin A deficient rats.

Induction of peroxisome proliferator responsive genes is thought to be mediated through binding of a peroxisome proliferator-activated receptor (PPAR) to specific peroxisome proliferator response elements in the upstream region of these genes. Binding of PPAR to the acyl-CoA oxidase promoter requires heterodimerization with the retinoid X receptor (RXR), and subsequent transactivation is strongest when ligands for both PPAR and RXR are present. Therefore, we hypothesized that depletion of ligand for the retinoid receptor would limit the induction of peroxisome proliferation in rats. Hepatic retinol content was reduced by more than 90% by feeding weanling rats a vitamin A deficient (VAD) diet for approximately 3 months. Nafenopin treatment for 7 days induced peroxisomal beta-oxidation 18-fold in VAD rats compared with 16-fold in rats fed a vitamin A sufficient (VAS) diet. Nafenopin induced microsomal laurate hydroxylase and mitochondrial beta-oxidation to comparable rates of specific activity in both VAD and VAS rats. However, the activities in VAD controls were significantly lower than in VAS controls, so the magnitude of the nafenopin-induced increases was greater in the VAD rats. Relative liver weights were increased nearly 2-fold in both VAS and VAD rats treated with nafenopin. Ultrastructural examination of the livers demonstrated that nafenopin increased the number and size of peroxisomes in both VAD and VAS rats. These data demonstrate that rats with severely depleted vitamin A stores remained responsive to the peroxisome proliferator nafenopin. Whether critical retinoid pools that supply RXR ligand (9-cis-retinoic acid) are spared in the vitamin A deficient rats remains to be determined.

Induction of peroxisomal beta-oxidation by nonsteroidal anti-inflammatory drugs.

Several chemical and pharmacologic agents have been identified as peroxisome proliferators in rodents. Most of these compounds contain a lipophilic backbone linked to an acid moiety, generally a carboxylate. Since ibuprofen and other nonsteroidal anti-inflammatory drugs share these structural characteristics, their effects on peroxisomal beta-oxidation were examined. Ibuprofen, flurbiprofen, and indomethacin caused dose-related increases in peroxisomal beta-oxidation in cultured rat hepatocytes. The dose-response for ibuprofen and flurbiprofen was roughly equivalent to that of clofibric acid, whereas indomethacin was less active. Ibuprofen and flurbiprofen are arylpropionic acids, which are structurally similar to the aryloxyisobutyric acid clofibric acid. Indomethacin differs structurally in that the acid substitution is on an indole ring. This structural difference may be responsible for the difference in activity. Ibuprofen and clofibric acid were also compared in vivo following 2-week dietary administration to rats. Ibuprofen increased relative liver weight and peroxisomal beta-oxidation and reduced serum lipids. Clofibric acid was more active than ibuprofen in vivo, particularly with respect to induction of peroxisomal beta-oxidation (16.8-fold vs 3-fold, respectively). The difference in activity of the two compounds in vivo was not consistent with the results in vitro. The disparity in peroxisomal activity of ibuprofen in the two test systems may be related to pharmacokinetic factors which are not present in vitro.

Effect of the peroxisome proliferator LY171883 on hepatocellular replication in female B6C3F1 mice.

LY171883 was shown to increase the incidence of hepatocellular carcinomas and other proliferative lesions in female B6C3F1 mice. This appeared to be unrelated to the induction of peroxisomal beta-oxidation. Experiments were conducted to determine the effect of dietary LY171883 for 7 or 94 days on hepatocellular replication using continuous 7-day infusion of bromodeoxyuridine. LY171883 caused a dose-related increase in hepatocyte replication during the first 7 days, with statistical significance in the two higher dose groups. There was no effect on hepatocyte replication after 94 days of treatment. Liver weight and peroxisomal beta-oxidation were increased in the two higher dose groups after 7 and 94 days, indicating there was not a general loss of hepatic responsiveness to LY171883. The data indicate that the hepatocarcinogenesis of LY171883 in female B6C3F1 mice is not associated with sustained replication in the general population of hepatocytes. It is possible that a mitogenic effect of LY171883 exerted on spontaneously initiated cells is involved in the development of the proliferative lesions; however, further work is needed to determine this.

Effect of the peroxisome proliferator LY171883 on triglyceride accumulation in rats fed a fat-free diet.

LY171883 is a leukotriene D4 antagonist that induces peroxisome proliferation in the rodent liver. Like many peroxisome-proliferating agents, it causes transient lipid accumulation and several other changes in hepatic lipid metabolism. The effect of LY171883 on lipid metabolism was studied further in rats maintained on a fat-free diet. Administration of a fat-free diet for 14 days caused a 5.6-fold increase in liver triglycerides associated with a 3.3-fold increase in fatty acid synthetase. Co-administration of 0.1% LY171883 increased liver triglycerides slightly, whereas 0.3% LY171883 prevented the accumulation of triglycerides. Furthermore, treatment with 0.3% LY171883 reversed the fatty liver in rats pretreated with the fat-free diet for 14 days. Fatty acid synthetase activity increased comparably in all treatment groups, indicating that 0.3% LY171883 did not prevent the lipogenic response to a fat-free diet. In rats treated with 0.3% LY171883, peroxisomal beta-oxidation increased 9.5-fold, mitochondrial beta-oxidation 4.8-fold, carnitine palmitoyltransferase I 1.9-fold, and plasma ketones 3-fold. In the 0.1% dose group the increases in these parameters were smaller. The data indicate that 0.3% LY171883 sufficiently increased mitochondrial and peroxisomal beta-oxidation such that fatty acids generated by lipogenesis were preferentially oxidized rather than esterified to triglycerides. In the 0.1% dose group oxidation was only mildly increased, and the excess fatty acids continued to be esterified.

Changes in hepatic lipid metabolism associated with lipid accumulation and its reversal in rats given the peroxisome proliferator LY171883.

Dietary administration of 0.05, 0.1, and 0.3% LY171883 to rats for 1 day caused a dose-related increase in hepatic triglycerides. When added to rat liver mitochondria in vitro, LY171883 caused competitive inhibition of carnitine palmitoyltransferase 1 (CPT-1), the rate-limiting enzyme for mitochondrial fatty acid oxidation. This effect appears to be involved in the lipid accumulation. The hepatic triglycerides in rats given 0.1% LY171883 increased progressively through 3 months of treatment. In contrast, hepatic triglycerides in high-dose rats returned to control levels by Day 3 and remained there throughout the study. The regression of the lipid corresponded with increases in hepatic peroxisomal beta-oxidation, mitochondrial beta-oxidation, and CPT-1 activity of up to 13-, 7-, and 3.2-fold, respectively. The 0.1% dose increased these parameters modestly compared to those of high-dose rats (2-, 3-, and 1.6-fold, respectively). Addition of LY171883 to mitochondria from rats given dietary treatment for 2 weeks inhibited CPT-I by the same percentage as in control mitochondria. In mid-dose rats, the induction of CPT-I was largely negated by LY171883 in vitro. Even with the inhibition, CPT-I activity in mitochondria from high-dose rats remained 2-fold higher than that in untreated controls. The data suggest that the induction of CPT-I in high-dose rats was sufficient to overcome the inhibitory action of LY171883. The increased oxidative capacity in peroxisomes and mitochondria led to the regression of the lipid in high-dose rats. The more modest increases in fatty acid oxidation in rats given 0.1% LY171883 were not sufficient to reverse the lipid accumulation.

Effects of chronic treatment with the leukotriene D4-antagonist compound LY171883 on B6C3F1 mice.

A 2-year toxicity/oncogenicity study was done to evaluate the potential effects of the leukotriene antagonist LY171883 in B6C3F1 mice. Dietary concentrations of LY171883 during the initial 7 months of the study were 0.0, 0.005, 0.015, or 0.05% but were increased to 0.0, 0.0075, 0.0225, or 0.075% during Months 7 through 24. The estimated average daily compound intake was 0.0, 7.3, 22.5, or 80.5 mg/kg for males and 0.0, 9.2, 27.5, or 95.9 mg/kg for females. Survival was not adversely affected by treatment, however, body weight of males and females in the high dose group was significantly lower than that of controls. The chronic toxicity was localized primarily to the liver. Liver weights were increased in males in the high dose group and in females in the mid and high dose groups. Microsomal p-nitroanisole-O-demethylase activity was increased in mid and high dose females. Hepatic peroxisomal beta-oxidation was increased approximately twofold in both sexes in the high dose group only. Centrilobular eosinophilic granular change of hepatocytes was a common histopathologic finding in male and female mice in the high dose group, with the incidence and severity being greater in females. An increased incidence of hepatocellular carcinomas was observed in female mice in the mid and high dose groups. The number of male mice in the high dose group with hepatocellular carcinomas was higher than that of controls but the change was not statistically significant. Hepatocellular adenomas were increased in females in the high dose group but not in males. All groups of treated females had increased nodular hepatocellular hyperplasia.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of ciprofibrate, bezafibrate, and LY171883 on peroxisomal beta-oxidation in cultured rat, dog, and rhesus monkey hepatocytes.

Cultured rat hepatocytes have been used extensively to study the mechanisms of chemically induced peroxisome proliferation. Hepatocytes from nonrodent species have been used on a limited scale to study interspecies differences in the response. Because of their importance in pharmaceutical safety assessment, we have developed a model to study the response of beagle dog and rhesus monkey hepatocytes to peroxisome proliferators. Treatment of the hepatocytes with peroxisome proliferators was begun after 20 hr in culture and continued for 72 hr. Untreated rat, dog, and monkey hepatocytes retained 62, 42, and 43% of their initial (20 hr) peroxisomal beta-oxidation activity throughout 92 hr of culture. Ciprofibrate, bezafibrate, and LY171883 caused a dose-related increase in beta-oxidation in rat hepatocytes to a maximum of 10-, 8-, and 5-fold, respectively. In dog and monkey hepatocytes the increases in beta-oxidation were less than 2-fold. Peroxisome morphology in dog and monkey hepatocytes appeared to be unchanged by the drugs. Morphometric analysis in monkey hepatocytes showed no increase in peroxisome volume fraction in response to the chemicals. Treatment of dog and monkey hepatocytes with dexamethasone and glucagon during the final 24 hr in culture caused a 4- to 6-fold increase in tyrosine aminotransferase activity. This induction is characteristic of the in vivo response. The small increase in beta-oxidation reflects the relative insensitivity of the dog and monkey liver to peroxisome proliferators in vivo rather than a loss of sensitivity during culture. Cultured hepatocytes from beagle dog and rhesus monkey may provide a model for studying the mechanisms underlying the interspecies differences. Such information would help clarify the relevance of rodent data in human risk assessment.

Effects of chronic treatment with the leukotriene D4 antagonist compound LY171883 on Fischer 344 rats and rhesus monkeys.

One-year toxicity studies were done to evaluate potential toxic effects associated with chronic exposure of rats and monkeys to the leukotriene antagonist LY171883. Rats were fed dietary doses of 0.0, 0.01, 0.03, or 0.1%, equivalent to approximately 0, 5, 15, or 50 mg/kg of body weight/day. Monkeys were given daily nasogastric gavage doses of 0, 30, 75, or 175 mg/kg of body weight. No treatment-related effects occurred in physical, behavioral, ocular, food consumption, or urinalysis parameters in either species. Mild dose-related hepatotoxicity occurred in rats given approximately 15 or 50 mg/kg of LY171883. The hepatotoxicity was characterized by liver enlargement associated with induction of hepatic peroxisomal beta-oxidation and microsomal drug metabolism. Male rats also had hepatocellular fatty change, centrilobular hypertrophy of hepatocytes, and increased levels of serum alanine transaminase and total bilirubin. Other effects in rats included minimal decreases in hematocrit values, decreases in serum triglycerides and cholesterol, and increased kidney weight. The monkeys tolerated daily oral doses of LY171883 up to 175 mg/kg with only minor increases in hepatic microsomal enzyme activity and slightly increased liver and kidney weights in males. No effects occurred in monkeys given 30 mg/kg. There was no induction of hepatic peroxisomal enzymes or pathologic abnormalities in monkeys treated with LY171883. The peroxisomal inductive effect was apparently a species-related effect separate from the pharmacologic activity of leukotriene antagonism.

Induction of peroxisomal beta-oxidation in the rat liver in vivo and in vitro by tetrazole-substituted acetophenones: structure-activity relationships.

LY171883, a leukotriene D4 antagonist in the tetrazole-substituted acetophenone structural class, previously was demonstrated to cause peroxisome proliferation in rodents. In the present studies, several analogs were tested to determine if there are structural requirements for the induction of peroxisomal beta-oxidation in the rat liver in vivo and in cultured rat hepatocytes. Liver weight and serum triglycerides also were measured in vivo. The increases in peroxisomal beta-oxidation caused by the tetrazole-substituted acetophenones in vivo ranged from negligible to greater than 17-fold and there was good agreement with the structure-activity relationships found in cultured hepatocytes. N-methylation of the acidic nitrogen of the tetrazole blocked the peroxisomal effects, indicating that the free acid was required for activity. The length of the alkyl chain linked to the tetrazole also influenced the activity of the compounds. However, the more important determinant of peroxisomal activity may be the spatial orientation of the acidic tetrazole with respect to the planar backbone of the molecule. The data indicate there is a target site for peroxisome proliferation in the liver that is able to distinguish between structurally similar analogs. This site appears to be distinct from the leukotriene receptor since both inducers and noninducers of peroxisomal beta-oxidation were shown previously to be potent leukotriene antagonists.

Inhibition of hepatic fatty acid oxidation by bezafibrate and bezafibroyl CoA.

The acute effect of the hypolipidemic agent bezafibrate on fatty acid oxidation was studied in rat hepatocytes and mitochondria. Bezafibrate caused a concentration-related inhibition of oleate oxidation in liver cells. In mitochondria bezafibrate inhibited the oxidation of palmitoyl CoA but had no effect on palmitoylcarnitine oxidation, suggesting the site of inhibition was the formation of the carnitine derivative. Bezafibrate and bezafibroyl CoA inhibited the overt carnitine palmitoyltransferase (I) in rat liver mitochondria with comparable potency but with distinct kinetics. The inhibition caused by bezafibrate was not prevented by omission of Mg++-ATP from the assay mixture, indicating activation of bezafibrate to bezafibroyl CoA was not required for inhibition. The data demonstrate that bezafibrate, like several other peroxisome proliferating agents, inhibits mitochondrial fatty acid oxidation in rat liver. The inhibition may be relevant to the mechanism of peroxisome proliferation.

Inhibition of hepatic fatty acid oxidation at carnitine palmitoyltransferase I by the peroxisome proliferator 2-hydroxy-3-propyl-4-[6-(tetrazol-5-yl) hexyloxy]acetophenone.

Recent studies suggest that the induction of peroxisomal beta-oxidation in rodents may represent an adaptive response to disturbances in hepatic lipid metabolism. The following studies were done to determine the effects of 2-hydroxy-3-propyl-4-[6-(tetrazol-5-yl)hexyloxy]acetophenone (4-THA), a tetrazole-substituted acetophenone which induces peroxisomal beta-oxidation in rodent liver, on fatty acid oxidation in vitro. In isolated hepatocytes, 4-THA inhibited the oxidation of oleate (C18:1) and decreased the mitochondrial redox state. The inhibition was more pronounced in the presence of 0.2 mM-oleate than with 0.5 mM, indicating the inhibition may be competitive. 4-THA had no effect on the oxidation of octanoate (C8:0), suggesting that the site of inhibition of oleate oxidation was the carnitine-dependent transport across the mitochondrial inner membrane. In rat liver mitochondria, 4-THA inhibited carnitine palmitoyltransferase I (CPT-I) competitively with respect to the substrate palmitoyl-CoA, increasing the apparent Km from 19 microM to 86 microM. The inhibition of CPT-I by 4-THA was independent of the concentration of the co-substrate carnitine. Whereas fasting attenuated the inhibition of CPT-I by malonyl-CoA, it did not diminish the inhibition by 4-THA. Inhibition of transferase activity by 4-THA and malonyl-CoA was attenuated in mitochondria which had been solubilized with octyl glucoside to expose the latent form of carnitine palmitoyltransferase (CPT-II), suggesting that the inhibition was specific for CPT-I. The specificity was further demonstrated in studies of mitochondrial beta-oxidation in which 4-THA inhibited the oxidation of palmitoyl-CoA but not palmitoylcarnitine. The results demonstrate that 4-THA inhibits fatty acid oxidation in rat liver in vitro at the site of transport across the mitochondrial inner membrane, CPT-I. Whether this disruption in mitochondrial oxidation is causally related to the induction of peroxisomal beta-oxidation is yet to be determined.

Hepatic peroxisomal changes induced by a tetrazole-substituted alkoxyacetophenone in rats and comparison with other species.

Previous work in this laboratory indicated that compound LY171883, a tetrazole-substituted alkoxyacetophenone with leukotriene D4 antagonist activity, caused dose-related hepatomegaly in rodents without other histological evidence of liver toxicity. In the present studies, administration of LY171883 at dietary concentrations of 0.25 or 0.50% to rats for 2 weeks increased peroxisomal beta-oxidation, catalase activity, and peroxisome volume fraction in the liver. The effects were dose-related and corresponded with increases in liver weight. Dietary concentrations of 0.05 and 0.1% LY171883 did not significantly alter peroxisome morphology, enzyme activity, or liver weight. Serum triglycerides were lowered equivalently by all four dietary concentrations of LY171883, indicating that the hypotriglyceridemia was dissociated from induction of peroxisomal beta-oxidation. The hepatic effects in rats reversed within 16 days after discontinuing treatment with LY171883. Liver weight and peroxisomal enzyme activities were increased in mice by LY171883 in a manner comparable to that observed in rats, whereas hamsters were less responsive. In guinea pigs there was a minor increase in beta-oxidation at a toxic dose of LY171883, but no change in catalase or liver weight. Neither hepatomegaly nor induction of peroxisomal enzymes occurred in beagle dogs or rhesus monkeys given LY171883. Since the hepatic effects of LY171883 in rats are not observed in higher species at a significant multiple of the anticipated clinical dose, it is unlikely that such effects will occur in humans.

Conditions influencing the induction of peroxisomal beta-oxidation in cultured rat hepatocytes.

Leibovitz L-15 medium was superior to RPM11640 in both maintenance and induction of peroxisomal beta-oxidation in cultured rat hepatocytes. Serum was not required for maintenance or induction of beta-oxidation. Cell plating density had only slight effects on maintenance and did not affect induction. Bezafibrate caused time- and dose-related increases in beta-oxidation, as well as increases in catalase, peroxisome volume fraction, and peroxisome number. This system maintained a greater percentage of control beta-oxidation than previously reported, and responded to several hypolipidemics with sizeable increases in activity. The flexibility of this system with respect to serum and cell-plating density facilitates simultaneous studies of cell parameters which are regulated by these variables.

Characterization of liver enlargement induced by compound LY171883 in rats.

Compound LY171883 caused dose-related and reversible hepatomegaly in male Fischer 344 rats. Histological examination revealed hepatocellular hypertrophy with no other evidence of liver disease. There were only minor changes in serum glucose, total bilirubin, alkaline phosphatase, and alanine transaminase which were generally unrelated to dose and dissociable from the hepatomegaly. Total liver DNA increased but the DNA concentration decreased, indicating that liver growth involved a combination of hypertrophy and hyperplasia. Total liver protein and RNA increased. Hepatic mitochondrial protein content increased but cytochrome oxidase activity was not changed. There were minor changes in mitochondrial respiratory parameters; however, all the values were in the normal range and there was no indication of mitochondrial toxicity. Microsomal protein, drug-metabolizing activity, and cytochrome P-450 increased, but glucose-6-phosphatase activity was not changed. The induction of drug-metabolizing enzymes and absence of toxicity were evidence that the hepatomegaly was an adaptation to an increased functional load in the liver. An increase in catalase activity suggested that the response may have also involved peroxisomes. In addition to rats, LY171883 administration caused hepatomegaly in mice and hamsters at daily exposures exceeding 100 mg/kg. The response was not observed in guinea pigs, beagle dogs, or rhesus monkeys given maximum tolerated doses, indicating LY171883-induced hepatomegaly is not a response common to all species. The doses required to elicit hepatomegaly greatly exceeded doses that produce pharmacological efficacy in animals and those that are expected to be used clinically. Since humans will not receive doses comparable to those given rodents, and considering that the primate species tested did not experience hepatomegaly, it is unlikely that the effect observed in rodents can be extrapolated to humans.